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Critical Factors for Successful Real-Time PCR - Gene

1. Tee coe ote herr er rer re rrr rer 13 3 4 1 0 p 0 9 COL 09 m p S n N S m 392 9 M 32 h D MM N NM 0 Cee rte Eroa meie Figure 43 Faster results without compromising sensitivity Expression of MYC a proto oncogene in human leukocytes was analyzed by realtime two step RT PCR Duplicate reactions were run on the Applied Biosystems 7500 Fast System using 10 fold cDNA dilutions 10 ng to 10 pg EY Fastcycling mode with the QuantiFast Kit gave similar C values to J standard cycling mode with the QuantiTect Kit Application data High sensitivity in fast real time RT PCR Performing real time RT PCR under fast cycling conditions can reduce the sensitivity of transcript detection The QuantiFast Probe PCR Kit enabled fast realtime RT PCR with much higher sensitivity than a kit from another supplier 1 0e 001 1 0e 000 Delta Rn 1 0e 001 1 23 458678 910111213141516 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 1 0e 002 Cycle Number Figure 44 Fast cycling using TaqMan assays without compromising performance Real time PCR was carried out on the ABI PRISM 7000 under fastcycling conditions using the QuantiFast Probe PCR Kit blue curves or a kit from Supplier Ey orange curves The template was twofold ser
2. Background Figure 10 PCR kinetics Reaction kinetics in PCR The Y axis I i is on a log scale 10 30 40 50 60 Cycle number A Same starting amount B Different starting amount dmo 0 gt 4 E ESTEE en 23 X X43572 MM 4 C 44 yale 30344 PNAKKENDNMASHOUMERMOH oye Figure 11 Problems associated with end point detection Two step RT PCR was carried out with the F same amount and J different amounts of template RNA The Y axis is on a linear scale M markers Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Quantification of target amounts 5 1 Quantification Target nucleic acids can be quantified using either absolute quantification or relative quantification Absolute quantification determines the absolute amount of target expressed as copy number or concentration whereas relative quantification determines the ratio between the amount of target and the amount of a control e g an endogenous reference molecule usually a suitable housekeeping gene This normalized value can then be used to compare for example differential gene expression in different samples 5 2 Absolute quantification Use of external standards enables the level of a gene to be given as an absolute copy number For gene expression analysis the most accurate standards are RNA molecules of known copy number or concentration Depending on the sequence and structure of the target and the efficiency of reverse trans
3. A Assay method Forskolin inhibition IC Traditional method 557 205 nM CV 0 37 FastLane Cell Multiplex Kit 564 217 nM CV 0 38 From 2 independent experiments each performed in duplicate Figure 48 Streamlined high throughput 18s rRNA screening without compromising performance UMR 106 cells were treated with four 10 fold Z SOST dilutions of forskolin 10 pM to 0 01 pM A Relative quantification of SOST expression was then carried out on the ABI PRISM 7900 using TaqMan assays for SOST and 18S rRNA 18S rRNA was used to normalize SOST expression ICs values for forskolin inhibition were determined for assays done using either the traditional method RNA purification followed by cDNA synthesis and duplex real time PCR or the FastLane Cell Multiplex Kit duplex real time one step RT PCR using cell lysates Amplification plots from multiplex reactions with the FastLane Cell Multiplex Kit are shown here for 1 experiment Data kindly provided by Angela Furrer and Hansjoerg Keller Bone amp Cartilage Unit Musculoskeletal Disease Area Novartis Institutes for 0 10 20 30 40 BioMedical Research Basel Switzerland Cycle 1 000 ARn 1 000 E 1 g How many copies of template can I detect This depends on the amplification reagents used and on successful primer design With QuantiTect and QuantiFast Kits lt 10 copies of template can be detected when the kits are used in combination
4. A QuantiFast SYBR Green PCR Kit Kit from Supplier fast cycling mode 48 minutes standard cycling mode 95 minutes z i 36 75 100 pg Figure 33 Sensitive detection of low copy numbers in fast real time RT PCR Tenfold serial dilutions of human leukocyte cDNA 100 ng to 100 pg were analyzed in duplicate on the Applied Biosystems 7500 Fast System using the QuantiTect Primer Assay for BCL2 and the indicated kits EY The QuantiFast SYBR Green PCR Kit provided significantly more sensitive detection of low copy numbers in half the time than H the kit from Supplier I Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Guidelines for successful real time PCR Application data High PCR efficiency for reliable relative quantification QuantiTect Primer Assays provide highly efficient amplification in SYBR Green based real time RT PCR This enabled reliable relative quantification of Nos2 and II1b expression in cultured rat aorta cells by the AAC method IIlb E 0 91 Figure 34 Reliable AAC based relative quantification of e Nos E 0 98 Nos2 and IIIb expression Real time two step RT PCR was 30 a Rnrl E 1 03 carried out on the ABI PRISM 7900 using the QuantiTect SYBR Green PCR Kit and QuantiTect Primer Assays The template was different dilutions of cDNA from cultured rat 20 ee aorta cells equivalent to 0 16 0 8 4 20 and 100 ng u a of RNA E Analysi
5. UDd LY SwWy d91 pup Yq utuni D 1 104 spnpoid NAOVIO o gt N Sen N S e 2 lt Q 8 2 4 S E _ e O e www qiagen com Useful links RNA somple technologies www qiagen com RNA PCR technologies www giagen com PCR RNAi solutions www giagen com siRNA GeneGlobe assays and siRNAs www qiagen com GeneGlobe ProductFinder www qgiagen com ProductFinder Gene expression analysis www giagen com geneXpression SYBR Green based real time PCR www giagen com SYBRGreen Multiple realtime PCR www qiagen com multiplex Fast real time PCR www giagen com fastPCR For up to date licensing information and productspecific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www qiagen com or can be requested from QIAGEN Technical Services or your local distributor Trademarks QIAGEN QlAamp QlAprep DNeasy FastLane GeneGlobe HotStarlaq MinElute Omniscript Q Bond QuantiFast Quantiscript QuantiTect REPLI g RNeasy Sensiscript QIAGEN Group ABI PRISM Applied Biosystems Primer Express StepOnePlus VIC Applera Corporation or its subsidiaries BHQ Black Hole Quencher Pulsar Quasar Biosearch Technologies Inc BLAST U S National Library of Medicine Bodipy Oregon Green SYBR Texas Red Molecular Probes Inc Cy GE Healthcare HRM
6. Fluorescence Threshold aia oe Figure 8 Typical amplification plot Amplification plots showing increases in fluorescence from 2 samples A and B Sample A contains a higher amount of starting template Baseline than sample B The Y axis is on a linear scale 0 10 20 30 40 Cycle number Baseline The baseline is the noise level in early cycles typically measured between cycles 3 and 15 where there is no detectable increase in fluorescence due to amplification products The number of cycles used to calculate the baseline can be changed and should be reduced if high template amounts are used or if the expression level of the target gene is high Figure 9A To set the baseline view the fluorescence data in the linear scale amplification plot Set the baseline so that growth of the amplification plot begins at a cycle number greater than the highest baseline cycle number Figure 9B The baseline needs to be set individually for each target sequence The average fluorescence value detected within the early cycles is subtracted from the fluorescence value obtained from amplification products Recent versions of software for various realtime cyclers allow automatic optimized baseline settings for individual samples 24 20 16 7 13 18 25 31 37 7 13 18 25 31 37 Cycle number Cycle number Figure 9 Correct baseline and threshold settings are important for accurate quantification EY Amplification product becomes detectab
7. H63D S65C heterozygote Figure 7 Sensitive detection of single base mutations Following realtime PCR using the QuantiTect Probe PCR Kit on the Rotor Gene 3000 amplification products were subjected to melting curve analysis Data kindly provided by T Kaiser Corbett Research r i i I j Australia 50 55 60 65 70 75 Temperature C Fluorescence dF dT Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Basic terms used in real time PCR 4 Basic terms used in real time PCR Before levels of nucleic acid target con be quantified in real time PCR the raw data must be analyzed and baseline and threshold values set When different probes are used in a single experiment e g when analyzing several genes in parallel or when using probes carrying different reporter dyes the baseline and threshold settings must be adjusted for each probe Furthermore analysis of different PCR products from a single experiment using SYBR Green detection requires baseline and threshold adjustments for each individual assay Basic terms used in data analysis are given below For more information on data analysis refer to the recommendations from the manufacturer of your real time cycler Data are displayed as sigmoidal shaped amplification plots when using a linear scale in which the fluorescence is plotted against the number of cycles Figure 8 m Sample A e Sample B Log lineor phose
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9. Germany www qiagen com Real Time PCR Brochure 07 2010 Methods in real time PCR 3 1 Two step and one step RT PCR Real time RT PCR can take place in a two step or one step reaction Figure and Table 2 With two step RT PCR the RNA is first reverse transcribed into cDNA using oligo dT primers random oligomers or gene specific primers An aliquot of the reverse transcription reaction is then added to the realtime PCR It is possible to choose between different types of RT primers depending on experimental needs Use of oligo dT primers or random oligomers for reverse transcription means that several different transcripts can be analyzed by PCR from a single RT reaction In addition precious RNA samples can be immediately transcribed into more stable cDNA for later use and long term storage In one step RT PCR also referred to as one tube RT PCR both reverse transcription and realtime PCR take place in the same tube with reverse transcription preceding PCR This is possible due to specialized reaction chemistries and cycling protocols see section 8 4 page 36 The fast procedure enables rapid processing of multiple samples and is easy to automate The reduced number of handling steps results in high reproducibility from sample to sample and minimizes the risk of contamination since less manipulation is required Add RT PCR master mix RNA template cDNA synthesis and PCR in 1 tube m Add RT Tr
10. The difference in C values was plotted against log template amount The difference in PCR efficiency was determined by calculating the slope of the line E Illustration showing how the AC value at one template amount is determined AC is the C value for HPRT subtracted from the C value for TNF Reference 1 Pfaffl M W Horgan G W and Dempfle L 2002 Relative expression software tool REST for group wise comparison and statistical analysis of relative expression results in realtime PCR Nucleic Acids Res 30 e36 www qiagen com Real Time PCR Brochure 07 2010 Quantification of target amounts Table 5 Data used to generate standard curves Template amount ng Log template amount C TNF C HPRT AC C TNF C HPRT 100 2 29 28 22 88 6 40 10 32 52 26 09 6 43 0 35 80 29 22 6 58 0 1 1 39 40 32 93 6 47 Table 6 Calculation of TNF expression levels in Jurkat cells using the AAC method C TNF C HPRT AC C TNF AAG AC Expression of TNF compared Sample average average C HPRT AC calibrator to calibrator 2 c Untreated 36 5 22 8 13 7 0 1 calibrator sample PMA 31 0 23 1 7 9 5 8 55 7 Jurkat cells were untreated or treated with PMA Total RNA was purified and after real time RT PCR the relative amounts of target and reference RNA were determined 5 3 8 Endogenous reference genes For relative quantification of gene expression it is important to choose a suitable
11. for reporter dyes commonly used in real time multiplex PCR E Choose suitable combinations of reporter dyes and quenchers that are compatible with multiplex analysis using the detection optics of your real time cycler for details refer to the instrument user manual for examples see Figures 54 and 55 For details on reporter dyes tested and recommended by QIAGEN see Table 15 E Use nonfluorescent quenchers e g use BHQ instead of TAMRA Triplex and 4 plex analysis may only be possible using nonfluorescent quenchers www qiagen com Real Time PCR Brochure 07 2010 Guidelines for successful multiplex real time PCR 10 2 10 3 Table 15 Combinations of reporter dyes for multiplex assays using QuantiFast and Quantilect Multiplex Kits Cycler Reference dye Dye 1 Dye 2t Dye 3t Dye 4 ABI PRISM 7700 ROX FAM HEX JOE VIC ABI PRISM 7000 and 7900 ROX FAM HEX JOE VIC Bodipy TMR NED Applied Biosystems 7300 StepOnePlus Applied Biosystems 7500 ROX FAM HEX JOE VIC Bodipy TMR NED Alexa Fluor 647 Cy5 iCycler iQ and iQ5 Not required FAM HEX JOE TET VIC Texas Red ROX Cy5 LightCycler 2 0 Not required FAM HEX JOE VIC Texas Red ROX Alexa Fluor 660 Bodipy 630 650 Pulsar 650 Mx3000P Mx3005P Not required FAM HEX JOE VIC Texas Red ROX Cy5 Rotor Gene 6000 Not required FAM HEX VIC ROX Quasar 705 Visit www qiagen com multiplex to view dye combinations for other cyclers including dye c
12. with optimal primer probe sets or primer sets such as QuantiTect Primer Assays g How can l avoid the formation of primer dimers We recommend using a hot start to PCR to prevent primer dimer formation In addition use a PCR buffer that promotes a high ratio of specific to nonspecific primer binding during the annealing step of each PCR cycle All QuantiTect and QuantiFast Kits provide a hot start and contain a PCR buffer specifically adapted for quantitative real time PCR and RT PCR In addition complementary sequences within and between the primers and probe should be avoided www qiagen com Real Time PCR Brochure 07 2010 Guidelines for successful real time PCR 2 l see several peaks in the melting curve of my SYBR Green experiment e g see Figure 38 page 44 What does that mean There can be several reasons for this In addition to the specific PCR product there may also be primer dimers in the reaction The T of primer dimers is generally lower than that of the specific PCR product A shoulder in the curve often above the T of the specific PCR product indicates nonspecific amplification Two distinct melting peaks could indicate the simultaneous amplification of cDNA and contaminating genomic DNA or the primers might have annealed to 2 different targets with identical primer binding sites e g 2 members of a homologous gene family What reaction volume should use QuantiTect Kits for real time PCR and
13. 5 4 4 Internal positive control An internal positive control can be used to test for the presence of PCR inhibitors A duplex reaction is carried out where the target sequence is amplified with 1 primer probe set and a control sequence i e the internal positive control is amplified with a different primer probe set The internal positive control should be at a high enough copy number for accurate detection If the internal positive control is detected but the target sequence is not then this indicates that the amplification reaction was successful and that the target sequence is absent or at too low a copy number to be detected g Why do replicates sometimes have different plateau heights The plateau phase of PCR is where the reaction is no longer in log linear growth and the height of the plateau indicates the yield of PCR product Identical amounts of starting template will not always result in identical yields of PCR product see Figure 11 page 15 Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Guidelines for preparation of template primers and probes 6 0 6 1 6 Guidelines for preparation of template primers and probes Success in realtime PCR and RT PCR depends on the purity and integrity of the template primers and probes used A template can be DNA or RNA purified from the biological sample being analyzed or a known amount of DNA or RNA to be used as a standard or positive control Primers
14. PPO 5 f PEE 2 000 E 1 j 0 01 ng cDNA f 1 ng cDNA j 6 000 E 1 pity FE 1 000 E 1 2 000 E 1 5 000 E 2 ff o 10 20 30 40 0 10 20 30 40 Cycle Cycle Figure 46 Sensitive two step RT PCR Reactions were run in triplicate on the ABI PRISM 7900 using 10 fold dilutions of human leukocyte cDNA 100 ng to 0 01 ng and a Primer Express designed TaqMan assay for IL8 a chemokine The QuantiFast Kit showed greater sensitivity than the kit from Supplier which was used according to the standard cycling protocol providing lower C values and transcript quantification from down to 0 01 ng cDNA 9 6 Real time RT PCR direct from cell lysates The ability to use cell lysates directly as template in real time RT PCR significantly streamlines the gene expression analysis workflow especially for high throughput analyses However several important factors need to be considered First of all RNA in the cell lysate must be protected from degradation and the upregulation and downregulation of mRNA transcripts immediately after cell lysis must be prevented Secondly the use of cell lysate as template in realtime two step or one step RT PCR must not compromise PCR specificity and sensitivity FastLane Kits are supplied with a lysis buffer that provides cell lysis with RNA stabilization Transcript representation is preserved to ensure accurate results in gene expression analysis The cell lysates also undergo a short reaction to remove genomic DNA This
15. Perform real time PCR RT PCR E Determine the C values for the standards and the samples of interest E Construct a standard curve for the endogenous reference gene by plotting C values Y axis against the log of template amount or dilution X axis E Calculate the amount of target and reference in the samples of interest using their C values and the standard curve E To calculate the normalized amount of target divide the amount of target by the amount of reference if replicate reactions were performed use the average value E Define the calibrator sample and compare the relative expression level of the target gene in the samples of interest by dividing the normalized target amounts by the value of the calibrator 5 3 6 Comparative method or AAC method of relative quantification An alternative approach is the comparative or AAC method which relies on direct comparison of C values The preparation of standard curves is only required to determine the amplification efficiencies of the target and endogenous reference genes in an initial experiment In all subsequent experiments no standard curve is required for quantification of the target sequence If amplification efficiencies are comparable amounts of target are simply calculated by using C values as described below First of all the AC value for each sample is determined by calculating the difference between the C value of the target gene and the C value of the endogenous referen
16. RT PCR and can be ordered at www giagen com GeneGlobe Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Quantification of target amounts Table 7 Housekeeping genes commonly used as endogenous references Gene symbol Relative expression level Gene Human Mouse Human Mouse 18S ribosomal RNA RRN18S Rn18s Actin beta ACTB Actb Glyceraldehyde 3 phosphate dehydrogenase GAPDH Gapdh Phosphoglycerate kinase 1 PGK1 Pgk1 Peptidylprolyl isomerase A PPIA Ppia Ribosomal protein L13a RPL13A Rpl13a Ribosomal protein large PO RPLPO Acidic ribosomal phosphoprotein PO Arbp Beta 2 microglobulin B2M B2m Tyrosine 3 monooxygenase tryptophan YWHAZ Ywhaz 5 monooxygenase activation protein zeta polypeptide Succinate dehydrogenase complex SDHA Sdha subunit A flavoprotein Fp Transferrin receptor TFRC Tfre Aminolevulinate delta synthase 1 ALAS1 Alas Glucuronidase beta GUSB Gusb Hydroxymethylbilane synthase HMBS Hmbs Hypoxanthine phosphoribosyltransferase 1 HPRT1 Hprtl TATA box binding protein TBP Tbp Tubulin beta TUBB Tubulin beta 4 Tubb4 indicates relative abundance of the transcripts 5 4 Controls 5 4 1 No template control A no emplate control NTC allows detection of contamination of the PCR reagents An NTC reaction contains all real time PCR components except the te
17. RT PCR have been optimized for a final reaction volume of 50 pl for 96 well plates or 20 pl for 384 well plates and LightCycler capillaries However volumes may be reduced to 20 pl and 10 yl respectively QuantiFast Kits for real time PCR and RT PCR have been optimized for a final reaction volume of 25 pl for 96 well plates 20 pl for LightCycler capillaries or 10 pl for 384 well plates We strongly recommend using the primer and probe concentrations reaction volumes and amount of starting template given in the QuantiTect and QuantiFast handbooks g Can l set up the reactions and store the plates containing all reaction components for later use With QuantiTect and QuantiFast Kits reaction plates can be stored for several hours at 4 C To avoid bleaching of probes or SYBR Green plates should be stored protected from light Reactions set up using QuantiTect and QuantiFast PCR Kits can be stored overnight at 20 C The effects of storage over an extended period cannot be predicted since factors such as type and quality of the probe quality of the template and the temperature consistency of the freezer may affect experimental results We do not recommend freezing reactions set up using QuantiTect and QuantiFast RT PCR Kits since the reverse transcriptases are sensitive to freezing when in an aqueous environment g How can l tell if have primer dimers in my reaction If using SYBR Green in quantitative PCR the cycling program may
18. SYBR Green Bodipy TMR 637 705 521 574 Bodipy 630 650 Alexa Fluor 680 EvaGreen 530 NED 575 640 702 es ii A An 400 450 500 550 600 650 700 Emission maximum nm Figure 4 Emission maximum of selected reporter dyes The emission maximum nm of selected reporter dyes are displayed in parentheses Emission maximum may vary depending on buffer conditions Other dyes with similar wavelengths may not be suitable for multiplex assays due to low fluorescence and or stability Real Time PCR Brochure 07 2010 www qiagen com Detection of PCR products in real time Table 1 Dyes commonly used for quantitative real time PCR Dye Excitation maximum nm Emission maximum nm Fluorescein 490 513 Oregon Green 492 Sy FAM 494 518 SYBR Green 494 521 TET 521 538 JOE 520 548 vice 538 552 Yakima Yellow 526 552 HEX 535 553 e 552 570 Bodipy TMR 544 574 NED 546 WS TAMRA 560 582 Gye 588 604 ROX 587 607 Texas Red 596 615 LightCycler Red 640 LC640 625 640 Bodipy 630 650 625 640 Alexa Fluor 647 650 666 Cy5 643 667 Alexa Fluor 660 663 690 Cy5 5 683 707 Emission spectra may vary depending on the buffer conditions My sample does not give a fluorescent signal How can decide whether this is because the PCR did not work or because the target is not expressed Use a control sample in which the gene of interest is definitely expressed or the target sequence is present PCR products th
19. SYBR Green binds all double stranded DNA molecules emitting a fluorescent signal of a defined wavelength on binding Figure 1 The excitation and emission maxima of SYBR Green are at 494 nm and 521 nm respectively allowing use of the dye with any real time cycler Detection takes place in the extension step of realtime PCR Signal intensity increases with increasing cycle number due to the accumulation of PCR product Use of SYBR Green enables analysis of many different targets without having to synthesize targetspecific labeled probes However nonspecific PCR products and primer dimers will also contribute to the fluorescent signal Therefore high PCR specificity is required when using SYBR Green 2 2 Fluorescently labeled sequence specific probes Fluorescently labeled probes provide a highly sensitive and specific method of detection as only the desired PCR product is detected However PCR specificity is also important when using sequence specific probes Amplification artifacts such as nonspecific PCR products and primer dimers may also be produced which can result in reduced yields of the desired PCR product Competition between the specific product and reaction artifacts for reaction components can compromise assay sensitivity and efficiency The following sections discuss different probe chemistries 2 2 1 TaqMan probes TaqMan probes are sequence specific oligonucleotide probes carrying a fluorophore and a quencher
20. and probes are DNA oligonucleotides which are typically purchased from a commercial supplier 6 1 Purification of nucleic acid templates Since PCR consists of multiple rounds of enzymatic reactions it is more sensitive to impurities such as proteins phenol chloroform salts and EDTA than single step enzyme catalyzed reactions Purity of nucleic acid templates is particularly important for realtime PCR since contaminants can interfere with fluorescence detection QIAGEN offers a complete range of nucleic acid purification systems that provide pure high quality templates for PCR and RT PCR These include QlAprep Kits for purification of plasmid DNA QlAamp and DNeasy Kits for purification of genomic DNA RNeasy Kits for purification of total RNA and the PAXgene Blood RNA System for stabilization and purification of RNA from blood Phenol and other contaminants can be efficiently removed from crude RNA preps using the RNeasy MinElute Cleanup Kit to clean up and concentrate RNA for sensitive assays Details about QIAGEN kits for nucleic acid purification can be found at www giagen com 6 1 1 Determining the integrity of RNA templates The integrity and size distribution of total RNA can be checked by denaturing agarose gel electrophoresis and ethidium bromide staining or by using a capillary electrophoresis system such as the QlAxcel www giagen com goto QlAxcel The respective ribosomal RNAs should appear as sharp bands or peaks
21. be optionally followed by melting curve analysis according to the instructions supplied with the realtime cycler Primer dimers will appear as a peak with a T usually between 70 C and 80 C that is less than the T of the specific product A detectable PCR product in the NTC usually indicates the presence of primer dimers If you see only 1 peak in all samples including the NTC you can run an agarose gel to check whether primer dimers have formed or to determine if the product is from nucleic acid contamination of the amplification reagents g How long should the amplicon be if am using SYBR Green detection For accurate quantification using SYBR Green the amplicon should be no longer than 150 bp In general the shorter the amplicon the higher the amplification efficiency g My amplification plots are hook shaped Why is that This phenomenon is sometimes observed when using nonhydrolyzoble probes such os FRET probes During the late phase of PCR where many PCR products have been generated there is strong competition between hybridization of the probe to the target strand and reassociation gt Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Guidelines for successful real time PCR of the 2 complementary product strands For some primer probe combinations reassociation occurs more quickly than probe hybridization toward the end of the PCR Therefore the yield of PCR product seems to decrease Figure 4
22. gene to use as a reference Table 7 The expression level of the reference gene must not vary under experimental conditions or in different states of the same tissue or cell line e g disease versus normal samples The expression level of the reference RNA should also be approximately the same as the RNA under study Reference RNA commonly used for relative quantification includes B actin B 2 microglobulin peptidylprolyl isomerase A and GAPDH mRNAs and also 18S rRNA actin mRNA is ubiquitously expressed and was one of the first RNAs to be used as a reference sequence However its transcription levels may vary and the presence of pseudogenes may mean that genomic DNA is detected during real time PCR leading to inaccuracies in quantification GAPDH is a housekeeping gene commonly used as a reference for quantification of gene expression GAPDH mRNA levels may vary between individuals at different stages of the cell cycle and following treatment with different drugs making GAPDH unsuitable as a reference in some systems As 18S rRNA is not an mRNA its expression levels in the cell may not accurately reflect the cellular mRNA population Therefore a combination of genes may provide the most reliable reference for quantification studies A comprehensive range of bioinformatically validated assays for housekeeping genes is available from QIAGEN These are QuantiTect Primer Assays which are primer sets for SYBR Green based real time
23. level of the target gene in the samples of interest by dividing the normalized target amounts by the value of the calibrator Table 3 Data used to generate standard curves Template amount ng Log template amount ng C IL8 C B2M 100 2 19 65 20 74 10 i 23 01 23 96 1 0 26 55 27 43 0 1 1 30 55 30 85 0 01 2 34 01 0 001 3 37 41 Slope 3 59 3 38 PCR efficiency 0 90 0 98 Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Quantification of target amounts A IL8 standard curve g B2M standard curve 40 35 35 30 30 O 25 25 20 20 1 5 T T T 1 1 5 T T T 1 4 2 0 2 2 0 1 2 Log ng total RNA Log ng total RNA Figure 15 Relative quantification with different PCR efficiencies Standard curves were generated for interleukin 8 IL8 and the housekeeping gene B 2 microglobulin B2M using total RNA from human leukocytes Real time RT PCR was performed using gene specific primers and probe with the QuantiTect Probe RT PCR Kit Table 4 Quantification and normalization of IL8 and B2M expression levels Amount of Amount of Normalized amount of Sample IL8 B2M C IL8 RNA ng B2M RNA ng IL8 RNA ng Ratio Untreated 37 01 18 83 1 40 x 10 346 4 05 x 10 1 0 calibrator sample PMA 29 43 18 59 PSO Os 417 492 x Ome 106 7 LTA 34 43 18 59 7 20 x 10 417 1 73 x 10 4 3 Jurkat cells were untreated or treated with either phorbol 12 myristate acetate PMA or lip
24. may not provide accurate results and may need to be adjusted C Save the multiplex reactions after amplification so that the PCR products can be checked on a gel if required A FAM channel B FAM channel default baseline settings cycle 3 15 adjusted baseline settings cycle 3 27 HEX channel E HEX channel default baseline settings cycle 3 15 adjusted baseline settings cycle 3 10 101 100 0 1 Threshold Cycle Caleutation Thresho M Use Treesboid 057 eam Figure 57 Importance of optimal baseline settings on data analysis Two targets were analyzed by real time singleplex PCR gray or realtime duplex PCR blue or red E The singleplex PCR and duplex PCR data for the FAM detected target are shown on the same graph The standard baseline settings cycles 3 15 have been applied However the singleplex PCR and duplex PCR data have different C values Note that the differences in C values may not be very obvious when inspecting the amplification plots in linear view not shown El The baseline settings for the data shown in EY have been adjusted cycles 3 27 the upper limit is set just before an increase in the fluorescent signal becomes detectable The singleplex PCR and duplex PCR data now show identical C values KA The singleplex PCR and duplex PCR data for the HEX detected target are shown on the same graph The standard baseline settings cycles 3 15 have been applied Since high template am
25. multiplex www qiagen com Real Time PCR Brochure 07 2010 Guidelines for successful multiplex real time PCR 10 4 Guidelines for evaluating the performance of a multiplex assay M Check the functionality of each set of primers and probe in individual PCR assays before combining the different sets in a multiplex PCR assay E Compare the performance of the multiplex PCR assay with the corresponding singleplex PCR assays Assay performance can be tested by for example assaying serial dilutions of a sample containing the target nucleic acids e g see Figure 54 In addition the dynamic range of the multiplex assay can be tested by for example making several dilutions of one target nucleic acid and keeping the concentration of the other target nucleic acid constant as template target nucleic acids cloned in a plasmid or prepared as a PCR product can be used E Check the multiplex PCR assay for linearity by performing reactions with 10 fold dilutions of template and check whether the C values obtained are similar to those obtained with the corresponding singleplex PCR assays A standard curve can be used to evaluate the linear range and the PCR efficiency of the assay e g see Figures 55 and 56 A c myc FAM B GAPDH HEX Amplification Curves Amplification Curves 4plex PCR a Aplex PCR Singleplex PCR ae Singleplex PCR s f sa f A f at Ga F 10 ng f HE f 0 1
26. nW 20 uoys PSIPINPIEN SIN HJ Dd x dii nW 2 euoyso4 XIU 1 sDui U ADd LY d ls suo x dii nw I qoud 2D up1ispj XIW J SDui U YOd Ly d ls suo aqold UD u 1O YAS 9D euoyso4 xiu JOJSOW U YOd Ly d ls suo u 1oO AGAS HA AN Yd LY xe dilnw Io luupno PEIN PN HJ ADd LY x dii nw joa_yuonD xiu JOJSOW u sYOd LY d ls suo HDA XOYON YOd x l du nw io liupno PIPPIN HEIN HI YDd x dii nw Peun XIU JO SOW U YOd LY dejs omy pu Yd x dii nw HA YDd LY eqoiq IS liupnio xiu J SDUI u sADd Ly d ls suo HI YDd 2901d Joa_yuoND xiu JOJSOW u YOd LY dajs omy pup YDd aqold HI AOdLY Y22015 YGAS poLyuoND xiu JOJSOW U YOd Ly d ls suo H Yd U99159 AAAS PELHUONH XIU 194S BU U 3Od 12I Aays omy pup YOd u 915 AJAS HJ A AOdLY x dii nw jsoquuons eqn lpipd s HJ AOd LY x di nWw jsoquuons xiu JOJSOW YOd Ly d ls suo HDL A YOd X dil nw jsoquuons eqn lpipd s HID ADd x dii nw Jsoquuons XIU JO SOW YDd LY dejs omy pup Yod x dii nw HJ DIA XOAF YOd LY 2901d IspojJliupnio qnl lpipd s HJ ADd LY 290d IspJliupnio xiu J sDUi ADd Ly d ls suo HDT PIA XON ADd Sql IspjJliupnio qnl lpipd s HID YDd 8qoid IspJilupnoO xiu J sDUI YOd LY dajs omy pup yod aqold HD ADd La YEPD AAAS POREM en eye YOd 1y d ls suO tH Yd 43910 AAAS SOIHUOND XIU AS45004 YDd 1y days omy pup YOd u 915 AJAS DI AP XOY n p d uo19349q qissod puawjoa SNN Burp pippupis e qissod jou juewyoed ONN Bui s04 Bojouypay
27. out The internal control can be either an endogenous gene that does not vary in expression between different samples e g a housekeeping gene see Table 7 page 24 or an exogenous nucleic acid For viral load monitoring the use of an exogenous nucleic acid as internal control allows the following parameters to be checked the success of sample preparation the absence of inhibitors and the success of PCR Multiplex analysis ensures high precision in relative gene quantification where the amount of a target gene is normalized to the amount of a control reference gene Quantification of multiple genes in a single reaction also reduces reagent costs conserves precious sample material and allows increased throughput Multiplex PCR and RT PCR are made possible by the use of sequence specific probes that are each labeled with a distinct fluorescent dye and an appropriate quencher moiety This means that the emission maxima of the dyes must be clearly separated and must not overlap with each other see Figure 4 on page 6 In addition reactions must be carried out on an appropriate real time cycler that supports multiplex analysis i e the excitation and detection of several non overlapping dyes in the same well or tube To view successful multiplex data from a wide range of real time cyclers visit www giagen com multiplex 3 3 Real time RT PCR using cell lysates Real time RT PCR is an ideal tool for cell assays that require accurate analys
28. sample but is usually a control e g an untreated sample or a sample from time zero of the experiment Positive control This is a control reaction using a known amount of template A positive control is usually used to check that the primer set or primer probe set works No template control NTC This is a control reaction that contains all essential components of the amplification reaction except the template This enables detection of contamination No RT control RNA preparations may contain residual genomic DNA which may be detected in realtime RT PCR if assays are not designed to detect and amplify RNA sequences only see section 9 7 page 52 DNA contamination can be detected by performing a no RT control reaction in which no reverse transcriptase is added Standard This is a sample of known concentration or copy number used to construct a standard curve Standard curve To generate a standard curve C values crossing points of different standard dilutions are plotted against the logarithm of input amount of standard material The standard curve is commonly generated using a dilution series of at least 5 different concentrations of the standard Each standard curve should be checked for validity with the value for the slope falling between 3 3 to 3 8 Standards are ideally measured in triplicate for each concentration Standards which give a slope differing greatly from these values should be discarded Efficiency and s
29. two step RT PCR 3 types of primers and mixtures thereof can be used for reverse transcription oligo dT primers 13 18mers random oligomers such as hexamers octamers or nonamers or gene specific primers Table 10 If oligo dT primers are used only mRNAs will be reverse transcribed starting from the poly A tail at the 3 end Random oligomers will enable reverse transcription from the entire RNA population including ribosomal RNA transfer RNA and small nuclear RNAs Since reverse transcription is initiated from several positions within the RNA molecule this will lead to relatively short CDNA molecules In comparison gene specific primers allow reverse transcription of a specific transcript Table 10 Suitability of primer types for RT PCR Application Recommended type of primer RT PCR of specific transcript Gene specific primer gives highest selectivity and only the RNA molecule of choice will be reverse transcribed RT PCR of long amplicon Oligo dT or gene specific primers RT PCR of an amplicon within long transcript Gene specific primers random oligomers or a mixture of oligo dT primers and random nonamers see section 8 1 1 are recommended so that cDNA covering the complete transcript is produced 8 1 1 Universal priming method for real time two step RT PCR A universal priming method for the RT step of real time two step RT PCR should allow amplification and detection of any PCR product regardless of tran
30. 0 Guidelines for successful reverse transcription Since RT PCR quantification of RNA is based on amplification of cDNAs the amount of cDNA produced by the reverse transcriptase must accurately represent original amounts to enable accurate quantification In comparison with other reverse transcriptases Omniscript Reverse Transcriptase a component of Quantiscript Reverse Transcriptase consistently gives higher sensitivity and a linear response Figure 28 A Low abundance transcript H High abundance transcript 2900 E 1 GIAGEN 1100 4 Supplier 2400 E 1 Supplier A 9000 E 1 1900 E 1 7000 E 1 c amp lt 1400 E 5000 E 1 9000 E 2 36063 kus 1000 E 1 1000 E 2 10 20 30 40 50 5 10 15 20 25 30 35 40 Cycle Figure 27 Higher sensitivity in real time two step RT PCR Real time two step RT PCR analysis of F TGFB2 low expression and E IL8 higher expression was carried out Total RNA was purified from human whole blood using the PAXgene Blood RNA system cDNA was then synthesized from 1 pg RNA using the QuantiTect Reverse Transcription Kit a kit from Supplier An or a kit from Supplier I Real time PCR was performed in duplicate on the ABI PRISM 7900 using the QuantiTect Probe PCR Kit and a gene expression assay for TGFB2 or IL8 The C values for TGFB2 were lowest with the QuantiTect Reverse Transcription Kit demonstrating that even low abundance genes can be efficien
31. 9 However for accurate quantification fluorescence data are measured during the log lineor phase of the reaction i e during the initial increase of fluorescence before this phenomenon occurs Amplification Curves Figure 49 Hook shaped amplification plot Amplification plot showing apparent decrease in yield of PCR product toward the end of PCR This phenomenon is typical for LightCycler instruments 1323 4 8 9 0313242141510137 1019221 222324 2 24 2 29 29 30 3 32 32 34 3 34 37 30 39 40 Cveles g The concentration of ROX dye in QuantiTect Probe Kits is too high for Auto C analysis on my Applied Biosystems 7500 What should I do We recommend setting the threshold manually For further details refer to the user manual supplied with your instrument My Bio Rad cycler iCycler iQ iQ5 or MyiQ requires use of fluorescein calibration dye Can still use QuantiTect and QuantiFast Kits Yes we recommend collecting external well factors with an external well factor plate containing fluorescein solution before starting your real time PCR run Our studies show that collecting external factors provides more reliable data with QuantiTect and QuantiFast Kits than collecting dynamic well factors which is achieved by spiking fluorescein into the PCR mix In addition collecting well factors is more convenient as the external well factor plate can be stored long term and reused many times Details on collect
32. A Polymerase which are modified forms of QIAGEN Taq DNA Polymerase provide a stringent hot start This hot start in combination with unique QIAGEN PCR Buffer enables high specificity in PCR Figure 37 A hot start also allows reverse transcription and PCR to be performed sequentially in one step RT PCR as the Taq DNA polymerase remains inactive during the RT step until activated at the start of PCR QIAGEN offers a wide range of reaction chemistries for real time PCR two step RT PCR and one step RT PCR with SYBR Green or probe detection QuantiTect Kits which include chemistries for multiplex analysis and QuantiFast Kits which provide fast cycling on any cycler to see the full range of kits refer to the selection guide on page 62 All QuantiTect Kits contain an application specific buffer based on QIAGEN PCR Buffer and HotStarlaq DNA Polymerase All QuantiFast Kits contain an adapted QIAGEN PCR Buffer for fast cycling as well as HotStarTaq Plus DNA Polymerase for a fast hot start Figure 37 Different hot start methods A 497 bp fragment was amplified from 50 copies of an HIV pol gene construct which had been added to 1 pg human genomic DNA Different hot start enzymes were employed HotStarlaq DNA Polymerase from QIAGEN hot start enzymes from Suppliers A and R Taq antibody mixture from Supplier or an enzyme with no hot start Arrow indicates the specific PCR product Equal volumes of the reaction were analyzed on a 2 agarose
33. Critical Factors for Successful Real Time PCR Sample amp Assay Technologies Contents 1 Introduction 2 Detection of PCR products in real time 2 1 SYBR Green 2 2 Fluorescently labeled sequence specific probes 3 Methods in real time PCR 3 1 Two step and one step RT PCR 3 2 Multiplex PCR and RT PCR 3 3 Real time RT PCR using cell lysates 3 4 Fast PCR and RT PCR 3 5 Whole genome amplification 3 6 Whole transcriptome amplification 4 Basic terms used in real time PCR 5 Quantification of target amounts 5 1 Quantification 5 2 Absolute quantification 5 3 Relative quantification 5 4 Controls 6 Guidelines for preparation of template primers and probes 6 1 Purification of nucleic acid templates 6 2 Handling and storing primers and probes 7 Guidelines for successful whole transcriptome amplification 7 1 WTA techniques 7 2 The importance of RNA quality 7 3 Working with degraded RNA 7 4 Amplification from low cell numbers www qiagen com A aA A Real Time PCR Brochure 07 2010 8 Guidelines for successful reverse transcription 8 1 Choice of RT primers 8 2 Effect of RT volume added to two step RT PCR 8 3 Reverse transcription conditions for two step RT PCR 8 4 Reverse transcription conditions for one step RT PCR 8 5 Removal of genomic DNA contamination 9 Guidelines for successful real time PCR 9 1 Primer design 9 2 Reaction chemistry 9 3 SYBR Green based detection in real time PCR 9 4 Probe based de
34. EER EERE SE 01234567 8 91081 42131615161718 192021 22232425262728293031 323334353637383940 Cycle Figure 41 Wide dynamic range and high sensitivity in two step RT PCR Duplicate reactions were performed on the Mastercycler ep realplex using 10 fold dilutions of human leukocyte cDNA and a Primer Express designed TaqMan assay for ubiquitin a regulatory protein The QuantiFast Probe PCR ROX Vial Kit provided accurate gene expression analysis from low to high template amounts with a PCR efficiency of 93 NTC no template control EN QuantiFast Probe PCR Kit ILIRN transcript Amplification Plot Total PCR time 57 min A 6 000 5 000 4 000 Van c ALS 3 000 rani 1 000 0 000 0 10 20 30 40 Cycle B Kit from Supplier A ILIRN transcript Amplification Plot Total PCR time 1h31 min f 3 000 2 500 2 000 1 500 1 000 0 500 Cycle Figure 40 Greater sensitivity in two step RT PCR Reactions were run in duplicate on the ABI PRISM 7900 using 10 fold dilutions of human leukocyte cDNA 100 ng to 0 01 ng and a TaqMan gene expression assay for ILIRN a cytokine The QuantiFast Kit showed greater sensitivity than the standard cycling kit from Supplier A which was used according to the standard cycling protocol providing much lower C values and transcript quantification from down to 10 pg cDNA V
35. PCR Kit enabled high PCR efficiencies of 95 for ssrRNA and 99 for DNA Pol Data kindly provided by Eric Prina Department of Parasitology and Mycology Institut Pasteur Paris France Application data Fast efficient real time PCR on any cycler Fast realtime PCR with SYBR Green detection was carried out on various cyclers using the QuantiFast SYBR Green PCR Kit and primers specific for RNase P A high PCR efficiency of around 100 was achieved with all cyclers tested Table 14 Fast real time PCR with high efficiency on various different cyclers Copy number Cycler 14 300 1430 143 14 3 1 43 Slope Efficiency R Applied Biosystems 7500 22 66 25 46 28 81 32 4 35 75 3 31 101 1 00 Fast iQ5 20 98 23 98 27 3i 30 02 34 05 3 219 104 0 99 LightCycler 1 2 21 54 24 52 27 97 31 36 3 349 99 0 99 Mx3000 20 31 23 28 26 78 0 230 241 3 314 100 0 99 The template was five 10 fold serial dilutions of human genomic DNA each analyzed in triplicate Data kindly provided by Karen McCaustland Centers for Disease Control and Prevention CDC Atlanta GA USA Presentation of these data does not imply endorsement by the CDC or any CDC employee www qiagen com Real Time PCR Brochure 07 2010 Guidelines for successful real time PCR m QuantiFast Probe PCR Kit B Kit from Supplier IL8 transcript IL8 transcript 1 800 s 4 000 E 1 1 400 3 000 E 1 j s 1 000
36. R The normalized value is determined for each sample and can be used for example to compare differential expression of a gene in different tissues or to compare gene expression between siRNA ransfected cells and untransfected cells However the expression level of the endogenous reference gene must not vary under different experimental conditions or in different states of the tissue e g stimulated versus unstimulated samples When gene expression levels are compared between samples the expression level of the target is referred to as being for example 100 fold higher in stimulated cells than in unstimulated cells The quantification procedure differs depending on whether the target and the endogenous reference gene are amplified with comparable or different efficiencies 5 3 1 Determining amplification efficiencies The amplification efficiency of 2 genes target A and target B can be compared by preparing a dilution series for both genes from a reference RNA or cDNA sample Each dilution series is then amplified in real time one step or two step RT PCR and the C values obtained are used to construct standard curves for target A and target B The amplification efficiency E for each target can be calculated according to the following equation E 10 _ 1 S slope of the standard curve To compare the amplification efficiencies of the 2 target sequences the C values of target A are subtracted from the C values of ta
37. Rotor Gene Corbett Research iCycler iQ Bio Rad Labortories Inc LightCycler TaqMan Roche Group Mx3000P Mx3005P Stratagene PAXgene PreAnalytiX GmbH Yakima Yellow Nanogen Inc Purchase of this product QuantiFast SYBR Green PCR Kit QuantiFast SYBR Green RT PCR Kit QuantiTect SYBR Green PCR Kit QuantiTect SYBR Green RT PCR Kit and FastLane Cell SYBR Green Kit is accompanied by a limited non transferable immunity from suit to use it with detection by a dsDNA binding dye as described in U S Patents Nos 5 994 056 and 6 171 785 and corresponding patent claims outside the United States for the purchaser s own internal research No real time apparatus or system patent rights or any other patent rights and no right to use this product for any other purpose are conveyed expressly by implication or by estoppel This product QuantiFast Probe PCR Kit QuantiFast Probe PCR ROX Vial Kit QuantiTect Probe PCR Kit QuantiTect Probe RT PCR Kit QuantiTect Multiplex PCR Kit QuantiTect Multiplex PCR NoROX Kit QuantiTect Multiplex RT PCR Kit QuantiTect Multiplex RT PCR NR Kit FastLane Cell Probe Kit FastLane Cell Multiplex Kit and FastLane Cell Multiplex NR Kit is an Authorized 5 Nuclease Core Kit without Licensed Probe Its purchase price includes a limited non transferable immunity from suit under certain patents owned by Roche Molecular Systems Inc or F Hoffmann La Roche Ltd for using only this amount of the product in t
38. The apparent ratio of 28S rRNA to 18S rRNA should be approximately 2 1 If the ribosomal bands or peaks of a specific sample are not sharp but appear as a smear towards smaller sized RNAs it is likely that the sample suffered major degradation either before or during RNA purification e g RNA integrity and quality is significantly reduced in formalin fixed paraffin embedded tissue samples 6 1 2 Determining the concentration and purity of nucleic acid templates The concentration of the DNA or RNA template should be determined by measuring the absorbance at 260 nm Az in a spectrophotometer Table 8 For accuracy absorbance readings at 260 nm should fall between 0 15 and 1 0 Alternatively RNA can be quantified using fluorescent dyes that bind specifically to RNA Measurement of the fluorescent signal on a fluorimeter enables RNA quantification Table 8 Spectrophotometric conversions for nucleic acid templates 1 A unit Concentration pg ml Double stranded DNA 50 Single stranded DNA 33 Single stranded RNA 40 Absorbance at 260 nm 1 1 cm detection path measurement in water www qiagen com Real Time PCR Brochure 07 2010 Guidelines for preparation of template primers and probes 6 1 6 2 Note that absorbance measurements at 260 nm cannot discriminate between DNA and RNA Depending on the method used for template preparation the purified DNA may be contaminated with RNA or the purified RNA may be contam
39. Transcription Kit which is specially designed to synthesize cDNA for use in realtime PCR contains an optimized primer mix consisting of a unique blend of oligo dT and random primers Figure 24 E Amplicon 2 kb from 3 end of template Oligo dT 1 uM Oligo dT N 1 uM 10 uM N 10 uM 20 25 30 Cycle number 35 40 CAP Amplicon kb from 3 end of template 3 0 Oligo dT 1 uM 2 5 Oligo dT N 20 1 uM 10 uM a N 10 uM a 15 1 0 OLD E ESENES I EEEE SS eee 0 T 1 15 20 25 30 35 40 Cycle number CaP AAA Figure 23 Effect of RT primer choice on RT PCR Two step RT PCR was carried out using the QuantiTect SYBR Green PCR Kit and the primer combinations shown The amplicon was E 2 kb from the 3 end or E kb from the 3 end of the template RNA Application data Highly sensitive real time two step RT PCR RT PCR 7 m QIAGEN I gett re Hh i ret 44 it ti z ji 34 i f 24 14 7 i a Pi O peepee WT 10 15 20 25 30 35 40 Cycle In contrast with other reverse transcriptases the QuantiTect Reverse Transcription Kit provides higher yields of cDNA from any transcript region allowing high sensitivity in real time two step Figure 24 Sensitive detection of a target at the 5 region of a 12 5 kb transcript Total RNA from mouse testis was reverse transcribed using the QuantiTect Revers
40. ady to use and is free of RNases and DNases Proper use of the buffer enables safe and accurate dilution of the small amounts of nucleic acids typically used as standards for analysis of viral nucleic acids Aliquots of diluted standards can be stored in QuantiTect Nucleic Acid Dilution Buffer at 20 C for up to months Repeated freezing and thawing should be avoided g My virus sample is very dilute Is real time PCR still possible QuantiTect Virus Kits are supplied with a highly concentrated 5x master mix This means that only one fifth of the final PCR volume is taken up by the PCR reagent allowing use of larger volumes of dilute viral template in the reaction Real Time PCR Brochure 07 2010 Sample amp Assay Technologies O BS Te OU Select HJ Od GUSEDAOJOY JO JOS UND JSO4YUOND D YIM Iy YNO PD PUOHO ey OSN uolipoljiund YNA InOUHA YDd Ly dajs OM SW JOY SHY SAJA JOO HUONG asn suoyODe x dil nuu Jo x d Buis u VNd JO pud YNY DJIA JO uoljo j p eAyIsuas uBiu 104 5 Hy uuoldiu59supi aJOYAA Jo2LHUOND y sn SJUNOWD YNy parwi Kaa wos SAIYID WN P aidad o l UOYdOsUDI SJ A9Y PALHUPNO Y sn OAOWS VNQGB YIM siseyjUds YNA 4S 104 sites sewiid apimewoueb peuBblsepeid skossy Jowlig JO LUDNS UHA pasn eq UDD 1 o S O SUEDAOJOY y PUD spy SueO 1oloq asn BuljoAd IspjJpij n 1oj 1A YN YOd x du
41. ansfer cDNA aliquots master mix to PCR master mix RNA template cDNA synthesis PCR Figure 6 Comparison of two step and one step RT PCR FE In one step RT PCR reverse transcription and PCR take place sequentially in the same tube E In two step RT PCR cDNA is synthesized in 1 tube and aliquots of the cDNA are transferred to other tubes for PCR Table 2 Advantages of different RT PCR procedures Procedure Advantages Two step RT PCR Multiple PCRs from a single RT reaction Flexibility with RT primer choice Enables long term storage of cDNA One step RT PCR Easy handling Fast procedure High reproducibility Low contamination risk Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Methods in real time PCR 3 2 Multiplex PCR and RT PCR In multiplex real time PCR several genomic DNA targets are quantified simultaneously in the same reaction Multiplex real time RT PCR is a similar method allowing simultaneous quantification of several RNA targets in the same reaction The procedure can be performed either as two step RT PCR or as one step RT PCR see section 3 1 Multiplex PCR and RT PCR offer many advantages for applications such as gene expression analysis viral load monitoring and genotyping The target gene s as well as an internal control are coamplitied in the same reaction eliminating the well to well variability that would occur if separate amplification reactions were carried
42. at span the region to be amplified in the real time experiment can also be used as a positive control Check by agarose gel electrophoresis that the amplification reaction was successful The quality of the starting template and the integrity of the reagents can be determined by amplifying a housekeeping gene such as GAPDH or HPRT or by amplifying the cloned target sequence either in the form of in vitro transcribed RNA or plasmid DNA g Do I need to calibrate my real time cycler in order to detect reporter dyes Some realtime cyclers require you to perform a calibration procedure for each reporter dye Check whether the reporter dyes you selected for your assays are part of the standard set of dyes already calibrated on your instrument If they are not perform a calibration procedure for each dye before using them for the first time For details about calibration refer to the user manual supplied with your cycler Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Methods in real time PCR 3 Methods in real time PCR DNA template e g genomic DNA or plasmid DNA can be directly used as starting template in real time PCR Real time PCR can be used to quantify genomic DNA for example in detection of bacterial DNA or GMOs and can also be used for qualitative analysis such as single nucleotide polymorphism SNP detection RNA template is used for analysis of gene expression levels or viral load of RNA viruses Rea
43. be parallel and the differences in C values of the target and the reference will not be constant when the template amounts are varied Figure 14 G e Target m Reference AC AC 2 AC 1 Figure 14 Different PCR efficiencies Typical standard curves showing amplification of 2 targets with different PCR efficiencies Log ng total RNA 5 3 3 Guidelines for relative quantification with different amplification efficiencies Choose an appropriate endogenous reference gene e g B 2 microglobulin or peptidylprolyl isomerase A whose expression level does not change under the experimental conditions or between different tissues Prepare a dilution series e g fivefold or tenfold dilutions of a cDNA or RNA control sample to construct standard curves for the target and reference Perform real time PCR RT PCR Determine the C values for the standards Table 3 and the samples of interest Construct standard curves for both the target and reference by plotting C values Y axis against the log of template amount or dilution X axis Figure 15 Calculate the amount of target and reference in the samples of interest using their C values and the corresponding standard curve Table 4 To calculate the normalized amount of target divide the amount of target by the amount of reference if replicate reactions were performed use the average value Define the calibrator sample and compare the relative expression
44. bition of realtime PCR Figure 25 We recommend testing dilutions of the RT reaction in real time PCR to check the linearity of the assay This helps to eliminate any inhibitory effects of the RT reaction mix that might affect accurate transcript quantification 8 3 Reverse transcription conditions for two step RT PCR The QuantiTect Reverse Transcription Kit provides efficient and sensitive reverse transcription of any template with Quantiscript Reverse Transcriptase which is a unique mix consisting of Omniscript and Sensiscript Reverse Transcriptases Omniscript Reverse Transriptase is designed for reverse transcription of RNA amounts greater than 50 ng and Sensiscript Reverse Transcriptase is optimized for use with very small amounts of RNA lt 50 ng This enzyme combination is compatible with a wide range of RNA template amounts from as little as 10 pg of RNA up to 1 pg of RNA Figure 26 RNA secondary structure can affect RT PCR in several ways Regions of RNA with complex secondary structure can cause the reverse transcriptase to stop or dissociate from the RNA template Table 11A Table 11 Effects of complex secondary structure on RT PCR RT effects Cause Effect on cDNA RT PCR result cDNA products missing primer binding site not amplified FE RT stops or dissociates at RNA region with complex secondary structure x Truncated cDNA products E RT skips RNA region with complex secondary structure Short
45. ce gene This is determined for each unknown sample as well as for the calibrator sample AC sample C target gene C reference gene AC calibrator C target gene C reference gene Next the AAC value for each sample is determined by subtracting the AC value of the calibrator from the AC value of the sample AAC AC sample AC calibrator If the PCR efficiencies of the target gene and endogenous reference gene are comparable the normalized level of target gene expression is calculated by using the formula Normalized target gene expression level in sample 2 see Table 6 Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Quantification of target amounts However if the PCR efficiency is not the same between the target gene and endogenous reference gene this method of quantification may lead to inaccurate estimation of gene expression levels The error is a function of the PCR efficiency and the cycle number and can be calculated according to the formula Error 2 1 E x 100 100 E efficiency of PCR n cycle number Therefore if the PCR efficiency is only 0 9 instead of 1 0 the resulting error at a threshold cycle of 25 will be 261 The calculated expression level will be 3 6 fold less than the actual value Note The AAC method should only be chosen if the PCR efficiency of the target gene and endogenous reference gene are the same or if the differen
46. ce in expression levels is sufficiently high to tolerate the resulting error However errors can be corrected by using efficiency corrected calculation programs such as the Relative Expression Software Tool REST 1 5 3 7 Guidelines for relative quantification using AAC method E Perform a validation experiment to determine the PCR efficiency for the target and reference as described in section 5 3 1 page 18 and shown in Figure 17 E Perform realtime RT PCR for the target and reference with RNA derived from different samples E Determine the AC value by subtracting the endogenous reference gene C value from the target gene C value for each sample Table 5 E Define the calibrator sample and determine the AAC value by subtracting the calibrator AC value from the AC value of each sample Table 6 E Calculate the normalized level of target expression relative to the calibrator by using the formula 2 c I AC slope 0 034 6 8 4 66 4 Mo AT S ias 6 4 4 4 62 4 ue Threshold 6 0 T T 1 I _1 0 1 2 Log ng total RNA Cycle number Figure 17 Comparison of PCR efficiency for TNF and HPRT genes F C values were determined for the tumor necrosis factor alpha TNF gene and the housekeeping gene hypoxanthine phosphoribosyl transferase HPRT using RNA purified from human leukocytes Realtime RT PCR was performed using the QuantiTect Probe RT PCR Kit and gene specific primers and probe
47. cies and can be easily ordered online at www giagen com GeneGlobe Figure 31 The assays have been optimized and validated to provide maximum sensitivity and a wide dynamic range Figures 32 34 for more data visit www giagen com SYBRGreen In contrast to other commercial assays primers are designed to cross exon exon boundaries where possible enabling amplification and detection of RNA sequences only Figure 51 page 53 This prevents coamplification of genomic DNA which can compromise assay sensitivity and efficiency by competition between the desired PCR product and the product derived from genomic DNA E GeneGlobe search page Enter your gene Select the Start the search Assay details page O Assay details page Hs_IL8_1_SG QuantiTect Primer Assay 200 OT00000322 Back to search results Last update of this data sheet Mar 01 2007 Nm 000584 1666 bp QIAGEN m 45103054457 HP GenomeWide siRNA m 45102654834 HP Validated siRNA m 49102654927 HP Validated aRNA m 48100012250 HP GenomeWide sRNA QY80680322 uai Ta er Pinar Seen 5 Eea EE EEE i ii Sun m mms 3 H u t t t t t H 1666 bp 0 200 400 600 800 1000 1200 1400 1600 Show p Transcript Selected product Hp Validated siRNA K w HP Genomevlide siRNA ssp Coding sequence F m Quanti Tecer Primer Assay Figure 31 Easy ordering of assays at GeneGlobe F Visit the GeneGlobe W
48. conjunction with the other components of this kit See U S Patent Nos 5 854 033 6 124 120 6 143 495 5 001 050 5 198 543 5 576 204 and related U S and foreign patents This product QuantiTect Primer Assays is compatible for use in the 5 nuclease process or the dsDNA binding dye processes covered by patents owned by Roche or owned by or licensed to Applera Corporation No license under these patents to practice the 5 nuclease process or the dsDNA binding dye processes are conveyed expressly or by implication to the purchaser by the purchase of this product The melting curve technology is covered by United States Patent No 5 871 908 and corresponding foreign patents owned by Evotec Biosystems GmbH and licensed to Roche Diagnostics GmbH The purchase of this product does not convey to the buyer any right under these patent rights to perform the melting curve technology claimed in those patents In particular the purchase of this product does not include nor carry any right or license to use develop or otherwise exploit the melting curve technology commercially and no rights are conveyed to the buyer to use the product or components of the product for any other purposes including without limitation provision of services to a third party generation of commercial databases research and development human diagnostics or veterinary diagnostics Roche Diagnostics GmbH reserves all rights under these patent rights For information on purchasing a l
49. cription only a proportion of the target RNA in the RNA sample will be reverse transcribed The cDNA generated during reverse transcription then serves as template in the subsequent real time PCR The use of RNA standards takes into account the variable efficiency of reverse transcription Brief guidelines for preparing RNA standards are given in section 5 2 1 page 17 A standard curve plot of C values crossing points of different standard dilutions against log of amount of standard is generated using a dilution series of at least 5 different concentrations of the standard Figure 12 The amount of unknown target should fall within the range tested Amplification of the standard dilution series and of the target sequence is carried out in separate wells The C values of the standard samples are determined Then the C value of the unknown sample is compared with the standard curve to determine the amount of target in the unknown sample Figure 12 40 4 m Standard sample 35 30 o 25 20 4 unknown 15 4 i i Initial concentration of unknown T Y T T 1 2 3 Log amount of standard Figure 12 Absolute quantification Typical standard curve showing determination of concentration of sample of interest It is important to select an appropriate standard for the type of nucleic acid to be quantified The copy number or concentration of the nucleic acids used as standards must be known In addition standa
50. d using varying amounts of template RNA as indicated Crossing point Cp or C values are shown for minute amounts of starting template or when detecting genes that are each reaction However even when primers and probes are designed using specialized expressed at a low level These problems can be minimized by using an appropriate reaction chemistry see section 9 2 page 42 Table 13 Primer design for real time PCR Sequence E Length of PCR product should ideally be less than 150 bp Avoid complementary sequences within and between primers and probes IE Avoid mismatches Avoid a 3 end T as this has a greater tolerance of mismatch Length 18 30 nucleotides GC content 40 60 Tm simplified T 2 C x A T 4 C x C G Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Guidelines for successful real time PCR 9 1 1 Assays for SYBR Green detection Primer design can be circumvented by using bioinformatically validated primer sets such as QuantiTect Primer Assays which are ready to use in SYBR Green based real time RT PCR on any realtime cycler Each assay comprising 2 gene specific primers as a lyophilized 10x mix provides high PCR efficiency and accurate quantification of as few as 10 copies of template when used in combination with Quantilect QuantiFast or FastLane Kits for SYBR Green based real time RT PCR Assays are available for any gene from human mouse rat and many other spe
51. e 2 probes bind their fluorophores come into close proximity allowing energy transfer from a donor fluorophore to an acceptor fluorophore Therefore fluorescence is detected during the annealing phase of PCR and is proportional to the amount of PCR product FRET probes usually carry dyes that are only compatible with LightCycler and Rotor Gene instruments As the FRET system uses 2 primers and 2 probes good design of the primers and probes is critical for successful results 2 2 3 Dyes used for fluorogenic probes in real time PCR For real time PCR with sequence specific probes various fluorescent dyes are available each with its own excitation and emission maxima Table 1 and Figure 4 The wide variety of dyes makes multiplex real time PCR possible detection of 2 or more amplicons in the same reaction provided the dyes are compatible with the excitation and detection capabilities of the real time cycler used and the emission spectra of the chosen dyes are sufficiently distinct from one another Therefore when carrying out multiplex PCR it is best practice to use dyes with the widest channel separation possible to avoid any potential signal crosstalk CAL Fluor Gold 540 544 JOE 548 Cy5 667 Yakima Yellow 548 Cy3 5 596 Alexa Fluor 647 VIC 552 ROX 607 666 HEX 553 CAL Fluor Red 610 Quasar 670 667 610 Texas Red 615 FAM 5 18 Alexa Fluor 660 Alexa Fluor 488 690 519 CAL Fluor Red 635 Quasar 705
52. e Transcription Kit or reverse transcriptases from Supplier and Supplier R Identical volumes of triplicate RT reactions were used in real time PCR on the LightCycler system to analyze a target located at the 5 region of the dystrophin gene about 12 5 kb upstream of the poly A site The error bars show the standard deviation for each set of triplicates Compared with the other 2 kits the QuantiTect Reverse Transcription Kit generated much higher amounts of cDNA indicated by the lower C values and provided greater reproducibility in realtime RT PCR indicated by the smaller error bars Data kindly provided by Dr Andrej Nikolai Spiess Department of Molecular Andrology University Hospital Hamburg Germany www qiagen com Real Time PCR Brochure 07 2010 Guidelines for successful reverse transcription 8 2 Effect of RT volume added to two step RT PCR In two step RT PCR the addition of the completed reverse transcription reaction to the subsequent amplification reaction transfers not only cDNA template but also salts dNTPs and RT enzyme The RT reaction buffer which has a different salt composition to that of the real time PCR buffer can adversely affect realtime PCR performance However if the RT reaction forms 10 or less of the final real time PCR volume performance will not be significantly affected Figure 25 Use of 3 pl of RT reaction in a 20 pl PCR i e 15 of the final volume can lead to significant inhi
53. e overall signal This effect reduces the range of the linear relationship between the number of cycles needed to detect the PCR product and the initial template amount Quantification of low copy templates is therefore often unreliable QuantiTect and QuantiFast SYBR Green Kits provide highly specific amplification allowing accurate quantification of just a few copies of template Figure 39 Specific and sensitive quantification is achieved with QuantiTect and QuantiFast SYBR Green Kits whatever the cycler used Amplification plots Copy number versus C value 40 38 7 0 99 36 34 32 30 28 2 24 T T T T T T 1 3 5 30 25 2 0 NS 1 0 05 0 Copy number log Cr value MECO A E Figure 39 Resolution of small differences in copy number The QuantiFast SYBR Green PCR Kit and the Mastercycler ep realplex were used to detect the Y chromosome specific single copy gene SRY in genomic DNA from a male donor EY Curves for 1000 down to 1 copy can be clearly distinguished from each other J A plot of copy number log versus C value demonstrates high linearity Visit www giagen com SYBRGreen to view application data for QuantiTect and QuantiFast SYBR Green Kits www qiagen com Real Time PCR Brochure 07 2010 Guidelines for successful real time PCR 9 4 Probe based detection in real time PCR Sequence specific probes provide a highly sensitive method of detection in realtime PCR and RT PCR In con
54. eb portal www qiagen com GeneGlobe enter the gene ID and species and start the search E After GeneGlobe finds the relevant assay you can click on o link to view details such as the position of the assay primers and amplicon size GeneGlobe can also search for assays for multiple genes Simply enter search terms into a single field or upload a list of search terms and start the search www qiagen com Real Time PCR Brochure 07 2010 Guidelines for successful real time PCR m SYBR Green based assay QIAGEN oa MAPKI C 25 1 J rur 16 C 30 7 40 amp 4 05 5 304 2 0 2 0 4 Fi 1 0 4 ee v 0 T T T T T T T 1 7 0 T T T t I I 5 10 15 20 25 30 35 40 60 65 70 75 80 85 90 95 Cycle Temperature C B Probe based assay Supplier A MAPKI C 29 5 2 07 l6 G 33 7 5 10 15 20 25 30 35 40 Cycle Figure 32 Superior sensitivity in real time RT PCR on the StepOnePlus MAPK1 a protein kinase and IL6 a cytokine in human leukocyte cDNA 1 ng were quantified in duplicate using EY QuantiTect Primer Assays and the QuantiTect SYBR Green PCR Kit or probe based assays and a real time PCR kit from Supplier A QuantiTect Primer Assays provided greater sensitivity than the probe based assays as demonstrated by the lower C values as well as high specificity as indicated by the single peaks in melting curve analysis
55. egrated genomic DNA removal www qiagen com Real Time PCR Brochure 07 2010 Guidelines for successful real time PCR 9 Guidelines for successful real time PCR PCR is both a thermodynamic and an enzymatic process Successful realtime PCR requires amplification and detection under optimal conditions and each reaction component can affect the result The annealing step is critical for high PCR specificity When primers anneal to the template with high specificity this leads to high yields of specific PCR products and increases the sensitivity of the amplification reaction However due to the high primer concentration in the reaction primers will also hybridize to noncomplementary sequences with mismatches If the primers anneal fo the template sequence with low specificity amplification of nonspecific PCR products and primer dimers may occur Competition in the amplification reaction between these artifacts and the desired PCR product may reduce the yield of the specific product thereby reducing the sensitivity and linear range of the real time reaction Low PCR specificity can significantly affect quantitative PCR particularly when using SYBR Green for detection As SYBR Green binds to any double stranded DNA sequence primer dimers and other nonspecific PCR products will generate a fluorescent signal This reduces the overall sensitivity of the assay and also leads to inaccurate quantification of the transcript of interest Factors critical
56. eliminates genomic DNA contamination in subsequent real time RT PCR and avoids the need to specially design primers or probes to prevent coamplification of genomic DNA sequences The cell lysates are prepared in only 12 minutes and can be used directly in real time two step or one step RT PCR using QuantiTect or QuantiFast technology without compromising performance Figures 47 and 48 L INPP5D FastLane 40 5 TAINPPSD RNeasy QuantiTect ESRF FastLane SRF RNeasy QuantiTect 307 8 204 Figure 47 Sensitive detection without RNA purification 5 Two low obundance transcripts INPP5D a phosphatase and SRF serum response factor were quantified in 3 different 10 cell lines by real time one step RT PCR The FastLane Cell SYBR Green Kit provided C values comparable to those achieved with the RNeasy Mini Kit and QuantiTect SYBR 0 Green RT PCR Kit Hela HepG2 Cos Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Guidelines for successful real time PCR Application data High throughput screening of gene expression Analysis of gene expression in multiple cell samples can be cumbersome due to the need to purify RNA from many samples The FastLane Cell Multiplex Kit allows multiplex real time one step RT PCR using cell lysates and provided similar results as multiplex realtime two step RT PCR in the analysis of forskolin inhibition of SOST expression in UMR 106 cells
57. emplate amounts that differ by twofold e g 10 ng and 5 ng the expected difference in Cr value would be 1 The data show that Cr values increased linearly by 1 C value with decrease in template dilution for both the E singleplex and El duplex reactions demonstrating the ability of the kit to precisely discriminate between small differences in template amount 10 5 Guidelines for programming the real time cycler E Be sure to activate the filters or detectors for the reporter dyes used in the multiplex PCR assay For details on setting up your real time cycler for multiplex PCR analysis refer to the instrument user manual E Follow the optimized protocols in the handbooks supplied with QuantiFast or QuantiTect Multiplex Kits It is important to follow the specified cycling conditions even for assays where the cycling conditions have already been established using a different kit or reagent 10 6 Guidelines for analyzing data from a multiplex assay E Optimal analysis settings i e baseline settings and threshold values for each reporter dye are a prerequisite for accurate quantification data LJ Adjust the analysis settings for every reporter dye channel in every run Figure 57 It is important to analyze the data for each channel separately www qiagen com Real Time PCR Brochure 07 2010 Guidelines for successful multiplex real time PCR L Note that the default analysis settings provided by the instrument software
58. en to avoid compromising the specificity and sensitivity of real time PCR Examples of fast PCR without compromising performance can be viewed for various different real time cyclers at www giagen com fastPCR 3 5 Whole genome amplification The small amount of genomic DNA in precious samples limits the number of real time PCR analyses that can be carried out This limitation can be overcome by whole genome amplification WGA a method which allows amplification of all genomic DNA targets in a sample Unbiased and accurate amplification of whole genomes can be achieved with Multiple Displacement Amplification MDA For details visit www giagen comn WGA 3 6 Whole transcriptome amplification Whole transcriptome amplification WTA allows amplification of entire transcriptomes from very small amounts of RNA enabling unlimited analyses by real time RT PCR WTA of RNA samples can be achieved by reverse transcription and cDNA ligation prior to MDA see page 28 Application data Single base detection using quenched FRET assays Quenched FRET assays are similar to FRET assays except that the decrease in energy of the donor fluorophore is measured instead of the increase in energy of the acceptor fluorophore FRET probes were designed to detect the single base mutations H63D and S65C The QuantiTect Probe PCR Kit enabled highly sensitive detection of wild type and mutant sequences Figure 7 Wild type H63D heterozygote
59. ened PCR products with internal deletions pa Shortened cDNA products with internal deletions Full length RT PCR GL products Full length cDNA products RT reads through Real Time PCR Brochure 07 2010 Sample amp Assay Technologies 3 5 2 5 15 Fluorescence 0 5 0 0 0 5 T T T T T 1 10 15 20 25 30 35 40 Cycle number Figure 25 Inhibition of real time PCR by addition of RT reaction Real time PCR 20 pl volume was carried out using plasmid DNA as template The volumes of RT reaction without template RNA indicated above were added to the PCR to determine their effect on amplification 4 000 3 500 3 000 2 500 ARn 2 000 1 500 1 000 HHHH 5 000 E 1 3 000 E 1 2 000 E 1 Derivative 1 000 E 1 0 000 70 74 78 82 86 90 Temperature C Figure 26 High specificity in real time two step RT PCR EN cDNA was synthesized from Hela cell RNA using the QuantiTect Reverse Transcription Kit Tenfold serial dilutions of cDNA 10 ng to 10 pg were then analyzed on the ABI PRISM 7900 using the QuantiTect SYBR Green PCR Kit and a QuantiTect Primer Assay for BCL2 B cell CLL lymphoma 2 EJ Melting curve analysis showed a single peak indicating high specificity Guidelines for successful reverse transcription The truncated cDNAs missing the downstream primer binding site are then not amplified durin
60. erse Transcription Kit which includes integrated removal of genomic DNA Figure 50 However if the intron exon boundaries of a gene are known appropriate primer and probe design can prevent amplification of sequences from genomic DNA Figure 51 9 7 2 Designing primers to avoid detection of genomic DNA The primer or probe can be designed so that one half hybridizes to the 3 end of one exon and the other half to the 5 end of the adjacent exon The primer or probe will therefore anneal to cDNA synthesized from spliced mRNAs but not to genomic DNA eliminating detection of contaminating DNA Alternatively RT PCR primers can be designed to flank a region that contains at least 1 intron Products amplified from cDNA no introns will be smaller than those amplified from genomic DNA containing introns If possible a target with very long introns should be selected the RNA target may then be preferentially amplified because of the higher PCR efficiency of this shorter PCR product without introns If genomic DNA is detected i e presence of amplification product in No RT control the template RNA should be treated with RNase free DNase Alternatively the primers should be redesigned to avoid amplification of genomic DNA Amplification Curves 13 is Figure 50 Effective genomic DNA removal for accurate i RT gt real time RT PCR Real time two step RT PCR using the QuantiTect Reverse Transcription Kit in combination wit
61. for high specificity in PCR include primer design and the reaction chemistry used 9 1 Primer design w an Prerequisites for successful real time PCR and RT PCR include design of optimal primer pairs for each reaction use of appropriate primer w fo concentration and correct storage of primer and probe solutions see N O section 6 2 page 27 For efficient amplification in real time PCR and RT PCR primers should be designed so that the size of the amplicon is ideally Crossing point N a os 626 bp 295 bp T T T T T of the reaction drop significantly with increasing amplicon size Figure 30 100ng 10ng Ing 100pg 10pg lpg lt 150 bp enabling comparison of amplification reactions for different targets Amplification efficiency represented by C values and sensitivity ol T 6 re s Template amount lt is particularly important to minimize nonspecific primer annealing so that high yields of specific PCR product are obtained The annealing step in PCR Figure 30 Improved PCR efficiency and sensitivity with shorter amplicons Quantitative real time one step RT PCR of is affected by both primer design and primer concentration Table 13 a 295 bp fragment and a 625 bp fragment of the B actin gene was carried out using the QuantiTect SYBR Green RT PCR Kit on the LightCycler system Reactions were software specificity can still be affected particularly when amplifying performe
62. for probes Threshold The threshold is adjusted to a value above the background and significantly below the plateau of an amplification plot It must be placed within the linear region of the amplification curve which represents the detectable log linear range of the PCR The threshold value should be set within the logarithmic amplification plot view to enable easy identification of the log linear phase of the PCR If several targets are used in the real time experiment the threshold must be set for each target Threshold cycle C or crossing point Cp The cycle at which the amplification plot crosses the threshold i e there is a significant detectable increase in fluorescence C can be a fractional number and allows calculation of the starting template amount see section 5 page 15 AC value The AC value describes the difference between the C value of the target gene and the C value of the corresponding endogenous reference gene such as a housekeeping gene and is used to normalize for the amount of template used AC C target gene C endogenous reference gene AAC value The AAC value describes the difference between the average AC value of the sample of interest e g stimulated cells and the average AC value of a reference sample e g unstimulated cells The reference sample is also known as the calibrator sample and all other samples will be normalized to this when performing relative quantificatio
63. g PCR Alternatively the reverse transcriptase can skip over looped out regions of RNA which are then excluded from the synthesized cDNA In the PCR step these cDNAs with internal deletions are amplified and appear as shortened PCR products Table 11B Ideally the reverse transcriptase should not be affected by RNA secondary structure Table 11C and should be capable of reverse transcribing any template without the need for reaction optimization Quantiscript Reverse Transcriptase s unique combination of Omniscript and Sensiscript Reverse Transcriptases has a particularly high affinity for RNA which means that the enzymes are closely associated with the template RNA This enables read through of templates with complex secondary structure or high GC content producing full length cDNA Figure 24 With high GC content the tight association of RNA DNA hybrids can interfere with primer binding during PCR and prevent DNA polymerases from progressing Table 12A RNase H an enzyme activity which is intrinsic to the Omniscript and Sensiscript Reverse Transcriptases of Quantiscript Reverse Transcriptase removes RNA in RNA DNA hybrids to allow primer binding and second strand DNA synthesis Table 12B RNase H digestion has been previously shown to improve RT PCR yield 1 and to be required for amplification of some sequences even as short as 157 bp 2 Table 12 Effects of high GC content on RT PCR PCR effects Cause Effect on PCR RT PCR re
64. g at least 20 bp upstream and downstream of the primer binding sites of the amplicons Copy number is calculated using the formula for plasmid DNA see above replacing plasmid length with the length of the PCR product Genomic DNA If the target of interest is present in only 1 copy per haploid genome and amplification of pseudogenes and or closely related sequences can be excluded genomic DNA can also be used as a DNA standard for absolute quantification The copy number of the target present in the genomic DNA can be directly calculated if the genome size of the organism is known For example the genome size haploid of Mus musculus is 2 7 x 10 bp a molecular weight of 1 78 x 10 Daltons 1 78 x 1012 g of genomic DNA corresponds to 6 022 x 10 copies of a single copy gene 1 pg of genomic DNA corresponds to 3 4 x 10 copies of a single copy gene Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Quantification of target amounts 5 3 Relative quantification In relative quantification the ratio between the amounts of a target gene and a control gene e g an endogenous reference gene present in all samples is determined This ratio is then compared between different samples In gene expression analysis housekeeping or maintenance genes are usually chosen as an endogenous reference The target and reference gene are amplified from the same sample either separately or in the same reaction duplex realtime PC
65. gel M markers 9 3 SYBR Green based detection in real time PCR Reduced specificity is often a major problem when using SYBR Green for quantitative real time PCR and RT PCR In comparison with kits from other suppliers QuantiTect and QuantiFast Kits for SYBR Green detection show improved PCR specificity when using SYBR Green Figure 38 gt Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Guidelines for successful real time PCR EN Agarose gel B Melting curves M R Q RT PCR Kit Supplier R QuantiTect SYBR Green RT PCR Kit 1 05 H 2 0 5 1 6 g P12 0 4 4 g 0 8 5 0 2 x 2 S 0 2 0 04 0 0 0 1 4r 0 2 68 70 75 80 85 90 68 70 75 80 85 90 Temperature C Temperature C Figure 38 Highly specific real time RT PCR RT PCR products were amplified from the B octin transcript in 1 ng Hela cell RNA on the LightCycler using an RT PCR kit from Supplier R R or the QuantiTect SYBR Green RT PCR Kit Q E Agarose gel electrophoresis and E melting curve analysis of the reactions showed a specific band and a single peak with the QuantiTect Kit indicating high specificity In contrast the kit from Supplier R gave a faint specific band and additional nonspecific bands as well as several peaks demonstrating low specificity M markers Low sensitivity can be a problem when using SYBR Green since fluorescence generated by nonspecific PCR products such as primer dimers contributes to th
66. h or the QuantiTect SYBR Green PCR Kit was carried out with be RT or without RT reverse transcriptase B actin specific y primers designed to detect both mRNA and genomic DNA o RT sequences were used Reactions were run in duplicate on Va the LightCycler 2 0 Control reactions with no template were mat also performed green The red flat RT plot indicates SS St ORR WE ECE YE Yya Y saw efficient removal of residual genomic DNA Oyos EN Forward primer spans an intron exon boundary Reverse primer spans an intron exon boundary Genomic DNA mRNA Genomic DNA mRNA al gt gt Exon Intron Exon Exon Exon Exon Intron Exon Exon Exon RT PCR RT PCR RT PCR RT PCR No product Product No product Product LU Figure 51 No coamplification of genomic DNA Primer design to eliminate signals from contaminating genomic DNA EX Forward primer crosses an intron exon boundary E Reverse primer crosses an intron exon boundary Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Guidelines for successful multiplex real time PCR 10 0 10 2 10 Guidelines for successful multiplex real time PCR Optimizing the conditions for multiplex real time PCR two step RT PCR and one step RT PCR can be tedious and time consuming Figure 52 Several factors need to be considered including the concentrations of the primers Mg Taq DNA polymerase and dNTPs as well as the composition of
67. he fast two step cycling i e denaturation followed by combined annealing extension with QuantiFast SYBR Green Kits provides the same sensitivity and specificity in realtime analysis as three step cycling i e denaturation annealing and extension with QuantiTect SYBR Green Kits The differences in annealing temperature between QuantiFast Kits and QuantiTect Kits are due to differences in buffer composition New Unparalleled speed and precision in real time PCR QIAGEN s realtime PCR cycler the Rotor Gene Q delivers outstanding optical and thermal performance through its unique rotary design When used in combination with dedicated Rotor Gene Kits for SYBR Green probe or multiplex detection extremely fast and precise quantification can be achieved The kits deliver reliable results using a ready to use master mix based on proven QIAGEN PCR buffers a V A balanced combination of ions promotes highly specific primer annealing Figure 35 page 42 eau while novel Q Bond greatly shortens cycling TE P 1 k v l SIF JN times Figure 42 page 46 A comprehensive range of Rotor Gene Kits and other QIAGEN kits for use with the Rotor Gene Q addresses all real time PCR and high resolution melting HRM applications including gene expression analysis genotyping pathogen detection gene scanning DNA methylation analysis and miRNA research For more information visit www giagen co
68. he method cannot be used to amplify RNAs substantially smaller than mRNA molecules such as tRNAs or miRNAs 7 4 Amplification from low cell numbers When carrying out WTA it is important to consider both the amount of starting material i e the number of cells or the amount of RNA and the copy number of the transcripts of interest Table 9 and Figure 21 show the relationship between the amount of starting material and transcript representation note that this is only a guide the number of transcripts per given amount of starting material can vary In starting material where the copy number of a transcript is 10 or less gt Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Guidelines for successful whole transcriptome amplification High copy transcripts Medium copy transcripts 4 Low copy transcripts Mosaic transcripts 10 000 000 1 000 000 100 000 10 000 1 000 100 10 0 01 0 1 10 RNA ng Copy number Figure 21 Transcript representation in different RNA amounts Relationship between amount of starting RNA and copy number of high copy medium copy low copy and mosaic transcripts Variation in C values PEPE CTE PONTO RN De Re Figure 22 Stochastic problems when analyzing a low copy transcript Eight replicate RNA samples 1 ng each were amplified using the QuantiTect Whole Transcriptome Kit Realtime PCR analysis of NF B transcript was then perfo
69. he practice of the 5 nuclease process solely for the purchaser s own internal research when used in conjunction with Licensed Probe No right under any other patent claims such as apparatus or system claims and no right to use this product for any other purpose is hereby granted expressly by implication or by estoppel Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA Purchase of this product QuantiFast SYBR Green RT PCR Kit QuantiTect SYBR Green RT PCR Kit QuantiTect Probe RT PCR Kit QuantiTect Multiplex RT PCR Kit QuantiTect Multiplex RT PCR NR Kit Fastlane Cell SYBR Green Kit FastLane Cell Probe Kit FastLane Cell Multiplex Kit and Fastlane Cell Multiplex NR Kit is accompanied by a limited non transferable license under RT and Reverse Transcription PCR patents owned by Roche Molecular Systems Inc and F Hoffmann La Roche Lid to use it for the purchaser s own internal research No real time patent rights of any kind no right under any other patent claims such as apparatus or system claims and no right to use this product for any other purpose is hereby granted expressly by implication or by estoppel The QuantiTect Whole Transcriptome Kit is for use only as licensed by Amersham Biosciences Corp part of GE Healthcare Bio Sciences and QIAGEN GmbH The Phi 29 DNA polymerase may not be re sold or used except in
70. ial dilutions of human skeletal muscle cDNA 100 ng to 3 13 ng Reactions were run in duplicate using a TaqMan assay for human NFKB1 nuclear factor of kappa light polypeptide gene enhancer in B cells 1 p105 Data kindly provided by Despina Constantin and Tim Constantin University of Nottingham Medical School Nottingham United Kingdom Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Guidelines for successful real time PCR Application data Sensitive parasite detection The formation of nonspecific products in SYBR Green based real time PCR reduces PCR specificity and sensitivity The QuantiFast SYBR Green PCR Kit overcomes this problem with a unique PCR buffer Two parasite DNA targets were amplified with high PCR efficiency and were sensitively detected A Target 1 ssRNA Target 2 DNA Pol a 2 c 2 gt 6 Slope 3 45 Slope 3 34 Intercept 37 95 Intercept 41 82 r 0 998 r 0 998 10 T T 1 T T T T 1 3 5 7 1 3 4 5 6 7 Log concentrations Log concentrations Figure 45 High PCR efficiences in parasite detection Fast real time PCR with SYBR Green detection was carried out on the LightCycler 1 5 using the QuantiFast SYBR Green PCR Kit The template was 6 or 5 dilutions of parasite genomic DNA from 5 ng to 0 5 pg or 0 05 pg Gene specific primers for ssrRNA 5S ribosomal RNA and DNA Pol DNA polymerase were used The standard curves show that the QuantiFast SYBR Green
71. icense to the patent rights for uses in conjunction with this product or to use the melting curve technology for other purposes please con tact Roche Diagnostics GmbH Patent Department Werk Penzberg Nonnenwald 2 82372 Penzberg Germany Certain specific embodiments of the process of multiplex PCR may be covered by patents of third parties in certain countries and may require a license Purchase of this product QuantiTect SYBR Green PCR Kit QuantiTect SYBR Green RT PCR Kit QuantiTect Probe PCR Kit QuantiTect Probe RT PCR Kit QuantiTect Multiplex PCR Kit QuantiTect Multiplex PCR NoROX Kit QuantiTect Multiplex RT PCR Kit and QuantiTect Multiplex RT PCR NR Kit is accompanied by a limited license under U S Patent Numbers 5 035 996 5 945 313 6 518 026 and 6 287 823 and corresponding foreign patents 2010 QIAGEN all rights reserved Real Time PCR Brochure 07 2010 Sample amp Assay Technologies www qiagen com Australia Orders 03 9840 9800 Fox 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 0086 21 3865 3865 Fax 0086 21 3865 3965 Technical 800 988 0325 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942
72. ils on choosing an appropriate combination of fluorophores for multiplex assays see section 10 2 page 54 or refer to the handbook supplied with the QuantiFast or QuantiTect Multiplex Kit 2 How many targets can l detect in 1 reaction vessel by multiplex PCR Up to 5 targets can be simultaneously detected in the same reaction vessel depending on the realtime cycler used For details refer to Table 15 page 55 or visit www giagen com multiplex www qiagen com Real Time PCR Brochure 07 2010 Guidelines for successful virus load quantitation 11 Guidelines for successful virus load quantitation When applying realtime PCR and RT PCR to detection of viral nucleic acids DNA and or RNA the inclusion of an internal positive control is often desired to rule out the possibility of false negatives In other words a multiplex reaction is carried out to quantify both the target viral nucleic acids as well as control nucleic acid Carrying out multiplex PCR and RT PCR also provides the advantage of detecting several viruses from the same sample simultaneously which saves times and conserves sample QuantiTect Virus Kits which are specially designed for sensitive detection of viral DNA and or RNA use similar multiplex PCR technology to QuantiTect Multiplex Kits Figure 58 allowing detection of up to 4 targets in a single reaction As little as 1 copy of viral DNA or RNA target can be detected either in multiplex reactions with an inter
73. in a 3 step cycling protocol with data acquisition during the extension step whereas TaqMan probes require a 2 step cycling protocol with data acquisition during the combined annealing extension step www qiagen com Real Time PCR Brochure 07 2010 Guidelines for successful real time PCR g Can make a master mix containing primers and all other reaction components and store it for later use The master mix supplied with QuantiTect or QuantiFast SYBR Green Kits can be mixed with primers and stored for more than 4 weeks at 20 C or 4 C If carrying out realtime one step RT PCR we do not recommend storing the primers master mix and RT mix as a single solution since reverse transcriptases are sensitive to freezing in an aqueous environment g Can I use QuantiTect Primer Assays to detect DNA No The assays are designed to detect RNA where possible The quality of my assays seems to decrease over time What could that be due to Make sure that primers and amplification reagents are stored correctly Avoid multiple freeze thaw cycles for primers Check the performance of your realtime instrument as some instruments require the halogen lamp to be frequently replaced Lasers must also be replaced occasionally g Can I use QuantiTect Primer Assays in fast real time PCR Yes QuantiTect Primer Assays are fully compatible with QuantiFast SYBR Green Kits which enable fast cycling in SYBR Green based real time PCR and RT PCR T
74. in real time PCR and RT PCR primers and probes should be purchased from an established oligonucleotide manufacturer Upon receipt the lyophilized primers and probes should be resuspended and their concentrations checked by spectrophotometry Lyophilized primers and probes should be dissolved in TE 10 mM Tris Cl 1 mM EDTA pH 8 0 to provide a stock solution of 100 pM and divided into small aliquots to avoid repeated freezing and thawing How much template can I use in the reaction and what is the maximum template volume Even when detecting low abundance targets we recommend using no more than 100 ng template RNA or cDNA Generally 1 100 ng template will be sufficient and for abundant transcripts as little as 1 pg can be used Template purity is important if large volumes of low concentration template are to be added to the reaction Using DNA or RNA purified with QIAGEN products the template can contribute up to 40 of the final reaction volume as long as the recommended template amounts are not exceeded If cDNA from an RT reaction is used as template the volume of undiluted RT reaction added should not exceed 10 of the final PCR volume g How does the method used to prepare the DNA or RNA template influence the real time PCR The purity of the starting template can have a tremendous effect on the quantification results since contaminants such as proteins phenol chloroform salts and EDTA can interfere with reverse transcription am
75. inated with DNA If this is the case the Ajo value will be too high and lead to inaccurate quantification The ratio between the absorbance values at 260 nm and 280 nm Ax6o Azeo gives an estimate of the purity of the DNA or RNA template As this ratio is influenced considerably by pH absorbance measurements should be made in 10 mM Tris Cl pH 7 5 In this buffer pure DNA has an Ajo Aogo ratio of 1 8 2 0 and pure RNA has an Ag o Areo ratio of 1 9 2 1 values of up to 2 3 are routinely obtained for pure RNA with some spectrophotometers Lower ratios indicate the presence of contaminants such as proteins 6 1 3 Storage of nucleic acid templates Purified DNA should be stored at 20 C or 70 C under slightly basic conditions e g Tris Cl pH 8 0 as acidic conditions can cause depurination of DNA Purified RNA should be stored at 20 C or 70 C in RNase free water Diluted solutions of nucleic acids e g dilution series used as standards should be stored in aliquots and thawed once only We recommend storage of aliquots in dedicated tubes that prevent adherence of nucleic acids to the tube walls Otherwise the concentration of nucleic acids in solution may be reduced QIAGEN offers QuantiTect Nucleic Acid Dilution Buffer supplied with QuantiTect Virus Kits which provides reliable dilution and storage of nucleic acid standards preventing adhesion on plastic surfaces 6 2 Handling and storing primers and probes For optimal results
76. ing well factors can be found in the instructions supplied with your Bio Rad cycler and also in our Technical Information www giagen com goto WellFactors 9 7 Genomic DNA in real time RT PCR Contaminating genomic DNA in RNA samples may also be detected in real time RT PCR leading to inaccurate quantification of the transcript of interest Depending on the method used for RNA purification and in particular what type of tissue or starting material was used for RNA purification genomic DNA can remain in the RNA sample Even if the primer or probe is designed to cross an exon exon boundary genomic DNA may still be detected due to the presence of pseudogenes Pseudogenes are nonfunctional copies of the respective gene that are highly homologous to their functional counterpart but usually lack introns Detection of DNA can be avoided by removing contaminating DNA with a DNase digestion step This may be necessary if pseudogenes exist if the gene of interest is a single exon gene if the organism being studied produces transcripts lacking introns e g bacteria or if sequence information is not available on intron exon boundaries www qiagen com Real Time PCR Brochure 07 2010 Guidelines for successful real time PCR 9 7 1 Eliminating genomic DNA contamination As an alternative to DNase digestion RNA can be purified using RNeasy Plus Kits which include gDNA Eliminator columns or cDNA can be synthesized using the QuantiTect Rev
77. ion with the QuantiTect Whole Transcriptome Kit preserves the gene expression profile of the RNA template E The experiment was repeated using a kit from Supplier R for whole transcriptome amplification Some targets were not uniformly amplified as some data points lay outside the dotted line that indicates 100 correlation 7 2 The importance of RNA quality The efficiency of WTA depends on the quality and quantity of the starting RNA template It is therefore important to use intact RNA High amounts of carrier RNA which may be used in procedures for purification of very low amounts of target RNA may affect the specific amplification of transcript sequences To ensure reproducible and efficient WTA we recommend purifying RNA template using silica membrane technology RNeasy Kits the PAXgene Blood RNA Kit and the QlAamp RNA Blood Mini Kit are based on this technology High amounts of carrier RNA gt 20 ng in the RNA purification procedure should be avoided However total RNA purified with carrier RNA using the RNeasy Micro Kit or RNeasy Plus Micro Kit performs well in WTA using the QuantiTect Whole Transcriptome Kit 7 3 Working with degraded RNA RNA from certain sources such as formalin fixed paraffin embedded FFPE tissue sections may be degraded and not be suitable for WTA For efficient RNA amplification with the QuantiTect Whole Transcriptome Kit RNA should not be heavily degraded i e RNA should be longer than 500 nucleotides T
78. is of gene expression for example in experiments to validate gene knockdown after transfection of siRNAs However high hroughput assays are difficult to achieve since the purification of RNA from large numbers of cultured cell samples involves both time and effort This bottleneck can be overcome by eliminating the RNA purification steps and using cell lysates directly in real time RT PCR However the method for cell lysis needs to be carefully optimized so that the lysates provide similar performance in realtime RT PCR as pure RNA templates The method should preserve the gene expression profile and also prevent cellular and buffer components from interfering with amplification and detection A reliable method for real time RT PCR direct from cells is presented at www giagen com FastLane www qiagen com Real Time PCR Brochure 07 2010 Methods in real time PCR 3 4 Fast PCR and RT PCR Most new real time cyclers are installed with thermal cycling modules that provide high ramping rates i e fast heating and cooling capacities This technology shortens the time to switch from one temperature to another allowing faster run times in real time PCR Further and more significant time savings can be achieved by reducing the duration of the denaturation annealing and extension steps in each PCR cycle as well as shortening the time required for activation of the hot start DNA polymerase When reducing these PCR parameters care should be tak
79. isit www giagen com fastPCR to view more application data for QuantiFast Probe Kits Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Guidelines for successful real time PCR 9 5 Real time PCR with fast cycling Fast real time PCR can be achieved by optimizing the reaction and cycling conditions of existing assays Such optimization includes reducing the DNA polymerase activation time shortening the duration of each PCR cycle combining the annealing and extension steps and shortening the RT step if carrying out one step RT PCR However changes to these parameters often reduce PCR sensitivity and increase the variability of quantification data QuantiFast Kits are supplied with a specially developed PCR buffer that allows a rapid hot start as well as a significant reduction of PCR cycling times HotStarlaq Plus DNA Polymerase which is inactive at ambient temperature requires only a brief 3 or 5 minute incubation at 95 C for activation The enzyme helps to increase PCR specificity by ensuring that no nonspecific products are formed during reaction setup and the initial denaturation step Reduced denaturation annealing and extension times are enabled by the patentpending additive Q Bond and other components of the PCR buffer Figure 42 High annealing specificity is maintained by a balanced combination of KCI and NH Cl in the buffer see section 9 2 1 page 42 For one step RT PCR an optimized mix of reverse transc
80. l time RT PCR is carried out where the RNA first needs to be transcribed into cDNA using a reverse transcriptase prior to PCR Reverse transcriptases are enzymes generally derived from RNA containing retroviruses Real time RT PCR can be either a two step or a one step procedure as described in section 3 1 Application data Using real time PCR for SNP genotyping SNP analysis involves the detection of single nucleotide changes using 2 probes labeled with different fluorophores One probe is specific for the wild type allele the other for the mutant allele Figure 5 Real time PCR is highly suited for the detection of small sequence differences such as SNPs and viral variants A ICAM5 E BCAC3 29 iets Deacrmnaten aN 7 pi gt As X asal Figure 5 Fast and reliable SNP genotyping Real time PCR with genomic DNA template and subsequent end point allelic discrimination was carried out on the Applied Biosystems 7500 Fast System using the QuantiFast Probe PCR ROX Vial Kit and TaqMan probes for detecting SNPs in EY ICAMS intercellular adhesion molecule and E BCAC3 29 The allelic discrimination plots clearly indicate the homozygotes for each allele Allele X or Allele Y and the heterozygotes A Both No template control is indicated by ll The PCR run time was about 35 minutes Data kindly provided by Peter Schuermann Department of Gynecology and Obstetrics Hannover Medical School Hannover
81. le within the baseline setting of cycles 6 to 15 and generates a wavy curve with the highest template amount E Setting the baseline within cycles 6 to 13 eliminates the wavy curve The threshold is set at the beginning of the detectable logtinear phase of amplification www qiagen com Real Time PCR Brochure 07 2010 Basic terms used in real time PCR Background This refers to nonspecific fluorescence in the reaction for example due to inefficient quenching of the fluorophore or the presence of large amounts of double stranded DNA template when using SYBR Green The background component of the signal is mathematically removed by the software algorithm of the real time cycler Reporter signal Fluorescent signal that is generated during real time PCR by either SYBR Green or a fluorescently labeled sequence specific probe Normalized reporter signal Rn This is the emission intensity of the reporter dye divided by the emission intensity of the passive reference dye measured in each cycle Passive reference dye On some real time cyclers the fluorescent dye ROX serves as an internal reference for normalization of the fluorescent signal It allows correction of wellto well variation due to pipetting inaccuracies well position and fluorescence fluctuations Its presence does not interfere with real time PCR assays since it is not involved in PCR and has an emission spectrum completely different from fluorescent dyes commonly used
82. lope The slope of a standard curve provides an indication of the efficiency of the realtime PCR A slope of 3 322 means that the PCR has an efficiency of 1 or 100 and the amount of PCR product doubles during each cycle A slope of less than 3 322 e g 3 8 is indicative of a PCR efficiency lt 1 Generally most amplification reactions do not reach 100 efficiency due to experimental limitations A slope of greater than 3 322 e g 3 0 indicates a PCR efficiency which appears to be greater than 100 This can occur when values are measured in the nonlinear phase of the reaction or it can indicate the presence of inhibitors in the reaction see section 8 2 page 33 The efficiency of a real time PCR assay can be calculated by analyzing a template dilution series plotting the C values against the log template amount and determining the slope of the resulting standard curve From the slope S efficiency can be calculated using the following formula PCR efficiency 1059 _ 1 x 100 Reference 1 Thellin O et al 1999 Housekeeping genes as internal standards use and limits J Biotechnol 75 291 www qiagen com Real Time PCR Brochure 07 2010 Quantification of target amounts 5 Quantification of target amounts With PCR minute amounts of starting template DNA or cDNA can be amplified enabling detection of a target sequence If PCR products are analyzed by end point analysis quantification is not possible as m
83. m goto Rotor GeneQ Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Guidelines for successful real time PCR 9 2 Reaction chemistry 9 2 1 Effect of cations on real time PCR specificity Cations especially Mg critically influence the melting behavior of DNA and therefore also affect the hybridization of primers to the target template K and Mg ions bind to the negatively charged phosphate groups on the backbone of the DNA This weakens the electrorepulsive forces between the target DNA and primer and stabilizes the primer template complex Figure 35 QIAGEN PCR Buffer provided with all QIAGEN PCR enzymes and kits has been developed to eliminate the need for optimization of individual primer template systems saving time and money The balanced combination of KCI and NH SO or NH Cl in the buffer promotes specific primer annealing without any need for optimization of Mg concentration maximizing yields of specific PCR product Figures 35 and 36 Kt cO Weak hyd bond TEN Primer s bases I mmm H gt j gt H ja Strong hydrogen bond Destabilization J between matching NH amp NH Ht H H bases 6 l Template aw Figure 35 NH and K cations in QIAGEN PCR Buffer increase specific primer annealing K binds to the phosphate groups P on the DNA backbone stabilizing the annealing of the primers to the template NH which exists both as the amm
84. moiety Figure 2 The fluorophore is attached at the 5 end of the probe and the quencher moiety is located at the 3 end During the combined annealing extension phase of PCR the probe is cleaved by the 5 3 exonuclease activity of Taq DNA polymerase separating the fluorophore and the quencher moiety This results in detectable fluorescence that is proportional to the amount of accumulated PCR product Examples of quencher moieties include TAMRA fluorescent dye and Black Hole Quencher BHQ nonfluorescent dyes Real Time PCR Brochure 07 2010 Sample amp Assay Technologies SYBR Green e Primer e ee 6 es y J 3 i e e o Primer oe 3 excitation emission Ti TATA LTA 3 Figure 1 SYBR Green principle Principle of SYBR Green based detection of PCR products in real time PCR Q fluorophore Quencher Cleaved nucleotides A excitation he G Primer Q A 3 5 Te a 9 Primer A L gt 3 5 Figure 2 TaqMan probe principle EX Both the TaqMan probe and the PCR primers anneal to the target sequence during the PCR annealing step The proximity of the quencher to the fluorophore strongly reduces the fluorescence emitted by the fluorophore EJ During the PCR extension step Taq DNA polymerase extends the primer When the enzyme reaches the probe its 5 3 exonuclease activity cleaves the fluorophore fr
85. mplate Detection of fluorescence in an NTC reaction indicates the presence of contaminating nucleic acids 5 4 2 Positive control A positive control may be necessary for example when amplifying a new target sequence to confirm whether the primer set or primer probe set works A positive control can be an absolute standard which is a nucleic acid template of known copy number that provides quantitative information Absolute standards such as a nucleic acid from an established cell line a plasmid containing cloned sequences or in vitro transcribed RNA are commercially available or can be generated in the lab A positive control can also be a known positive sample which is usually a substitute for an absolute standard and used only to test for the presence or absence of a target 5 4 3 No RT control A no RT control where real time RT PCR is carried out without reverse transcriptase should be included when performing gene expression analysis www qiagen com Real Time PCR Brochure 07 2010 Quantification of target amounts For viral load monitoring a no RT control may be necessary depending on the sample type and the life cycle of the virus species detected Since reverse transcription cannot take place a no RT control reaction allows detection of contaminating DNA such as DNA from viral sequences integrated into the host genome Contaminating DNA in RNA samples can be removed by DNase treatment before starting RT PCR
86. n AAC average AC sample of interest average AC reference sample For more information on AC and AAC see section 5 page 15 Endogenous reference gene This is a gene whose expression level should not differ between samples such as a housekeeping gene 1 Comparing the C value of a target gene with that of the endogenous reference gene allows normalization of the expression level of the target gene to the amount of input RNA or cDNA see AC value The exact amount of template in the reaction is not determined An endogenous reference gene corrects for possible RNA degradation or presence of inhibitors in the RNA sample and for variation in RNA content reverse transcription efficiency nucleic acid recovery and sample handling See page 24 for a list of housekeeping genes gt Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Basic terms used in real time PCR Internal control This is a control sequence that is amplified in the same reaction as the target sequence and detected with a different probe i e duplex PCR is carried out An internal control is often used to rule out failure of amplification in cases where the target sequence is not detected Calibrator sample This is a reference sample used in relative quantification e g RNA purified from a cell line or tissue to which all other samples are compared to determine the relative expression level of a gene The calibrator sample can be any
87. nal control or in single target amplification reactions Multiplex assays without any optimization steps are achieved through QuantiTect Virus PCR Buffer which contains an optimized combination of KCl and NH Cl as well as synthetic factor MP for effective primer annealing and HotStarlaq Plus DNA Polymerase which provides a stringent hot start with a short activation time 4 QuantiTect Virus Kit E Supplier A Supplier Figure 58 Improved detection of low amounts of viral RNA ND a compared with other real time kits Viral RNA was diluted in serial fivefold dilutions and amplified in duplex with an g oe internal control using the QuantiTect Virus Kit or kits from 30 Suppliers A and The QuantiTect Virus Kit provided much Oo higher sensitivity than the other reagents tested enabling 20 reliable analysis of unknown samples ND Not detected 10 ioe fter 50 PCR cycles T T T T T T 3 23 16 09 02 0 5 1 2 Template dilution How can I safely dilute RNA and DNA standards to very low concentrations We recommend using QuontiTect Nucleic Acid Dilution Buffer supplied with QuantiTect Virus Kits to dilute nucleic acids RNA or DNA used to generate standard curves or used as positive controls in real time PCR or RT PCR The buffer stabilizes RNA and DNA standards during dilution and reaction setup and prevents loss of nucleic acids on plastic surfaces such as tubes or pipet tips The buffer is re
88. ng 3 10 ng W lng _ 2 se T s QL ra MA nasti Te 8 w 0 54 9 m Z N N 2 X S N N N Q G lt P Q 0 Az w 5 9 ZX 2 N 5 9 NX N NM N 35 G G one Opes HSP89 Texas Red E H28S Alexa Fluor 660 Amplification Curves Amplification Curves i Aplex PCR p 4plex PCR E Singleplex PCR a EEA Singleplex x E ERA w PCR f 77 f a 1 T 13 im 10ng 1a lng Ww Ji U J 0 1 ng x J j J LL BESS EES OS SS ORAS SSS UR e nas tear ney oe are oe ss N Figure 54 Comparable amplification in 4 plex PCR and singleplex PCRs 4 plex real time one step RT PCR was performed on the LightCycler 2 0 using the QuantiTect Multiplex RT PCR NR Kit and TaqMan probes The template was 10 1 or 0 1 ng of total RNA purified from K562 cells EY c myc a proto oncogene was detected using a FAM labeled probe E GAPDH glyceraldehyde 3 phosphate dehydrogenase was detected using a HEX labeled probe I HSP89 a heat shock protein was detected using a Texas Red labeled probe BJ H28S 28S rRNA was detected using an Alexa Fluor 660 labeled probe For comparison the targets were also quantified by singleplex real time one step RT PCR black curves Curves for 4 plex and singleplex PCRs overlap showing comparable amplification i e equivalent C values Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Guidelines for successf
89. om the probe The fluorescent signal from the free fluorophore is measured The signal is proportional to the amount of accumulated PCR product Detection of PCR products in real time Fluorophore donor Z Fluorophore acceptor excitation p J Primer y 5 excitation transfer emission Primer g ee r 3 85 excitotion k we Primer 3 5 Figure 3 FRET probe principle EY When not bound to the target sequence no fluorescent signal from the acceptor fluorophore is detected E During the PCR annealing step both FRET probes hybridize to the target sequence This brings the donor and acceptor fluorophores into close proximity allowing energy transfer between the fluorophores and resulting in a fluorescent signal from the acceptor fluorophore that is detected The amount of signal is proportional to the amount of target sequence and is measured in real time to allow quantification of the amount of target sequence During the extension step of PCR the probes are displaced from the target sequence and the acceptor fluorophore is no longer able to generate a fluorescent signal Alexa Fluor 350 442 Marina Blue 460 i 2 2 2 FRET probes PCR with fluorescence resonance energy transfer FRET probes such as LightCycler hybridization probes uses 2 labeled oligonucleotide probes that bind to the PCR product in a head to tail fashion Figure 3 When th
90. ombinations for 5 plex PCR t Preferably select Dye 1 for the least abundant target Dye 2 for the second least abundant target and Dyes 3 4 for the most abundant targets 10 3 Choice of PCR buffer The amplification of multiple targets in the same reaction presents the All QIAGEN multiplex kits Other methods challenge of ensuring that all targets are amplified with the same high P efficiency If the reaction contains targets that vary significantly in Follow kit protocol Develop strategy abundance the more abundant targets may be amplified more efficiently than the less abundant targets resulting in inaccurate quantification data Time A typical strategy to overcome this problem is to determine the limiting primer concentration for the more abundant targets However this may require numerous optimization steps until the multiplex assay works satisfactorily Figure 52 Successful results without QuantiTect Multiplex Kits provide a ready to run solution for multiplex optimization real time PCR and RT PCR The supplied master mix which contains Successful results novel QuantiTect Multiplex PCR Buffer and HotStarlaq DNA Polymerase only after lengthy optimization eliminates the need for optimization of reaction and cycling conditions enabling successful results at the first attempt All PCR products in a Figure 52 Prerequisites for success in real time multiplex PCR Developing multiplex assays often requires length
91. onium ion and as ammonia under thermal cycling conditions can interact with the hydrogen bonds between the bases B destabilizing principally the weak hydrogen bonds at mismatched bases The combined effect of the 2 cations maintains the high ratio of specific to nonspecific primer template binding over a wide temperature range Stabilization Buffer with NH Buffer without NH M 15 44 M 15 m mM Mg Figure 36 Specific primer annealing over a range of Mg concentrations PCR amplification of the single copy human prion protein gene was carried out at increasing Mg concentrations from 1 5 mM to 4 mM PCR was performed using either QIAGEN PCR Buffer and QIAGEN Tag DNA Polymerase Buffer with NH or PCR buffer and Tag DNA polymerase from Supplier P Buffer without NH M markers 0 75 kb www qiagen com Real Time PCR Brochure 07 2010 Guidelines for successful real time PCR 9 2 2 Effect of a hot start on real time PCR specificity Real time PCR specificity can also be increased by using a hot start where an inactive DNA polymerase is activated at the start of PCR by incubation at a high temperature A hot start increases PCR specificity because it prevents the formation of primer dimers and nonspecific products during reaction setup and the initial heating step After a hot start these PCR artifacts are also absent in every PCR cycle HotStarlaq DNA Polymerase and HotStarlaq Plus DN
92. ost reactions will already have reached the plateau phase of amplification During this phase no significant increase in the amount of PCR product takes place Figure 10 This is mainly due to depletion of PCR components and renaturation of PCR product strands caused by the high concentration of end products which prevents further primer annealing If identical template amounts are used this may not necessarily result in identical yields of PCR products Figure 11A Equally if different amounts of template are used the yield of PCR products may be similar making quantification impossible Figure 11B Real time PCR overcomes this problem by determining the actual amount of PCR product present at a given cycle indicated by the intensity of fluorescence The fluorescence generated by SYBR Green or fluorescently labeled probes is indicative of the amount of PCR product in the reaction provided the reaction is in the log linear or so called exponential phase see Figure 10 and provided that amplification proceeds with a comparable efficiency for all samples By selecting the threshold within the log linear phase for all samples it is possible to calculate the actual amount of initial starting molecules since the fluorescence intensity is directly proportional to the amount of PCR product in the exponential phase Exponential phase Plateau phase 2 i 5i c 2 2 i b Lgi i oS 1 0 3 a 2 5 s f
93. oteichoic acid ITA Total RNA was purified and after real time RT PCR the relative amounts of target and reference RNA were determined using the appropriate standard curve see Figure 15 5 3 4 Comparable amplification efficiencies If the amplification efficiencies of the target gene and the endogenous reference gene are comparable one standard curve for the reference gene is sufficient The differences in C values of the target and the reference will be constant when the amounts of template are varied Figure 16 The amounts of target and reference in an unknown sample are calculated by comparing the C values with the standard curve for the reference gene as described below Target m Reference AC 1 AC 2 C Figure 16 Same PCR efficiencies Typical standard curves showing amplification of 2 targets with similar PCR efficiencies Log ng total RNA www qiagen com Real Time PCR Brochure 07 2010 Quantification of target amounts 5 3 5 Guidelines for relative quantification with comparable amplification efficiencies E Choose an appropriate endogenous reference gene e g B 2 microglobulin or peptidylprolyl isomerase A whose expression level does not change under the experimental conditions or between different tissues E Prepare a dilution series e g fivefold or tenfold dilutions of a cDNA or RNA control sample to construct a standard curve for the endogenous reference gene only E
94. ounts were used detectable amplification appears already within the baseline cycles Due to software algorithms wavy curves are displayed PJ The baseline settings for the data shown in have been adjusted cycles 3 10 so that no detectable amplification appears within the baseline cycles The wavy curves are now eliminated Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Guidelines for successful multiplex real time PCR 21 quantify my target gene and control gene in separate reactions and have already optimized the primer probe concentrations Can use these concentrations when analyzing both genes in a multiplex assay Yes If using QuantiFast or QuantiTect Multiplex Kits you can continue to use the same primer probe concentrations However be sure to follow the cycling conditions specified in the handbook supplied with the kits B I have already designed primer probe sets for my target gene and control gene Do need to redesign them if want to analyze both genes in a multiplex assay First of all check whether the primers and probes are complementary to each other If they are not redesign of the primer probe sets is not necessary if combining them for use with QuantiFast or QuantiTect Multiplex Kits However it may be necessary to label the probes with new fluorophores Each probe should be labeled with a distinct fluorophore and detected via a particular channel of your realtime cycler For deta
95. plification and fluorescence detection For sensitive applications we recommend spiking a sample to be purified with an exogenous control such as an in vitro transcript before the purification procedure The amplification efficiency can then be compared to a positive control to monitor for the presence of inhibitors acquired during the sample preparation process gt Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Guidelines for successful whole transcriptome amplification 7 Guidelines for successful whole transcriptome amplification When only nonogrom amounts of an RNA sample are available the number of real time RT PCR analyses that can be performed is limited This problem can be resolved by whole transcriptome amplification WTA With this technology all mRNA transcripts in an RNA sample are replicated to provide microgram amounts of cDNA template enough for unlimited real time PCR analysis and stable archiving 7 1 WTA techniques To ensure reliable results in real time PCR it is essential that the WTA method provides unbiased and accurate amplification of the whole transcriptome This means that the sequence and the relative abundance of each transcript should be preserved after WTA otherwise false results in gene expression analysis will occur The method used by the QuantiTect Whole Transcriptome Kit to prepare amplified cDNA dedicated for use in real time PCR consists of the following steps reverse tran
96. rds should have the following features E Primer and probe binding sites identical to the target to be quantified E Sequence between primer binding sites identical or highly similar to the target sequence E Sequences upstream and downstream from the amplified sequence identical or similar to the natural target E Equivalent amplification efficiencies of standard and target molecules www qiagen com Real Time PCR Brochure 07 2010 Quantification of target amounts 5 2 1 RNA standards for absolute quantification RNA standards can be created by cloning part or all of the transcript of interest into a standard cloning vector The insert can be generated by RT PCR from total RNA or mRNA or by PCR from cDNA The cloning vector must contain an RNA polymerase promoter such as T7 SP6 or T3 Ensure that in vitro transcription of the insert leads to generation of the sense transcript After in vitro transcription plasmid DNA must be removed completely with RNase free DNase since residual plasmid DNA will lead to errors in spectrophotometric determination of RNA concentration and will also serve as a template in the subsequent PCR Furthermore ensure that the RNA used as a standard does not contain any degradation products or aberrant transcripts by checking that it migrates as a single band in gel or capillary electrophoresis After determination of RNA concentration by spectrophotometry see section 6 1 2 page 26 the copy n
97. rget B The difference in C values is then plotted against the logarithm of the template amount Figure 13 If the slope of the resulting straight line is lt 0 1 amplification efficiencies are comparable 7 0 Figure 13 Efficiency comparison C values were determined re AC slope 0 034 for the tumor necrosis factor alpha TNF gene and the housekeeping gene hypoxanthine phosphoribosyl transferase 6 6 HPRT using RNA purified from human leukocytes Realtime Y 7777777 77 fercc RT PCR was performed using the QuantiTect Probe RT PCR gii Kit and gene specific primers and probe The difference in C values was plotted against log template amount The difference in PCR efficiency was determined by calculating the slope of the line AC Log ng total RNA 5 3 2 Different amplification efficiencies Amplification efficiencies of the target gene and the endogenous reference gene are usually different since efficiency of primer annealing GC content of the sequences to be amplified and PCR product size usually vary between the 2 genes In this case a standard curve needs to be prepared for the target gene as well as for the endogenous reference gene for example using total RNA prepared from a reference cell line calibrator or reference sample www qiagen com Real Time PCR Brochure 07 2010 Quantification of target amounts Due to differences in PCR efficiency the resulting standard curves will not
98. riptases enables cDNA synthesis in just 10 minutes Fast real time PCR is achieved without compromising specificity and sensitivity Figures 43 46 and Table 14 for more data visit www qiagen com fastPCR A Standard cycling 3 4 DULNI N DUAN 3 B Fast cycling 4 V a gt 5 3 Highly specific annealing DD N 3 4 QBond molecule Taq DNA polymerase N N Template DNA Primer Figure 42 Fast primer annealing Q Bond dramatically increases the binding affinity of DNA polymerase to single stranded DNA enabling faster annealing and extension F Under standard cycling conditions denaturation annealing and extension usually occur as a 3 step process where the template is first denatured followed by primer annealing to form a binary complex and then polymerase binding to form a tertiary complex EJ With Q Bond in the reaction this 3 step process becomes a faster 2 step process where the template is first denatured and the tertiary complex is then formed by the simultaneous binding of primer and polymerase allowing extension to start within seconds Other components of the buffer support the melting of double stranded DNA allowing reduced denaturation and extension times www qiagen com Real Time PCR Brochure 07 2010 Guidelines for successful real time PCR A QuantiFast SYBR Green PCR Kit B QuantiTect SYBR Green PCR Kit fast cycling mode standard cycling mode Beta Pe ena te
99. rmed using 10 ng amplified cDNA and the QuantiFast Probe PCR Kit The resulting C values were in the range of 30 32 5 This significant variation occurred because the stochastic variation in low copy NF B transcript in the replicate RNA samples is amplified resulting in widely differing amounts of NFkB cDNA highlighted in bold in Table 9 stochastic problems will occur i e the unequal distribution of a very low number of transcripts in a highly dilute solution This may result in underrepresentation of the low copy transcript at the start of WTA Special consideration should be given to mosaic transcripts which are derived from genes that are expressed only in a subset of cells in tissues Since these transcripts are not present in every cell they will not be accurately represented in low amounts of starting material i e 1 10 cells Reliable WTA depends on the copy number of the transcripts If the recommended amount of starting material i e 10 ng intact RNA is amplified with the QuantiTect Whole Transcriptome Kit all transcripts will be accurately represented after amplification 10 ng of RNA corresponds to about 500 cells and even low copy transcripts are well represented in this RNA amount Using lower amounts of RNA or a very limited number of cells means that the starting material could have a partial representation or an absence of low copy transcripts Figure 22 provides an example Table 9 Transcript representa
100. s of Nos2 nitric oxide synthase 2 m inducible expression Analysis of II1b interleukin 1 beta 10 expression IA Calculation of PCR efficiencies for Nos2 II1b and Rnr1 ribosomal 18s and 28s RNA gene 1 The comparable PCR efficiencies for target and control genes 0 ft j enabled reliable AAC based relative quantification data 10 100 1000 not shown Data kindly provided by Miriam Cortese Log cDNA Heinrich Heine University D sseldorf Germany g Can I use QuantiTect Primer Assays for validation of siRNA experiments Yes We have seen that 24 hours or later after transfection of cells with siRNA from QIAGEN the reduction in transcript level could be reliably detected with QuantiTect Primer Assays both with assays encompassing the cleavage site as well as with assays located up and downstream of it For more information read QIAGEN News weekly article e14 September 2006 at www giagen com literature giagennews Can I use QuantiTect Primer Assays and assays using TaqMan probes in the same run Yes this may be possible when using QuantiFast Kits as the kits allow a combined annealing extension step for both types of assay Please follow the cycling recommendations in the handbook supplied with the QuantiFast SYBR Green Kit you are using However if using QuantiTect Kits QuantiTect Primer Assays and TaqMan assays should be run separately With QuantiTect Kits QuantiTect Primer Assays are used
101. script length and amplicon position and achieve this with high sensitivity and reproducibility To determine the optimal primers for reverse transcription 2 regions within a 10 kb transcript were analyzed by real time two step RT PCR Each amplicon was approximately 150 bp in length For the amplicon 2 kb away from the 3 end of the transcript the lowest C value was obtained using either oligo dT primers alone or a mixture of oligo dT primers and random nonamers Figure 23A However when the amplicon was 6 kb away from the 3 end of the transcript random nonamers provided the lowest C value closely followed by the mixture of oligo dT primers and random nonamers Figure 23B The use of oligo dT primers alone resulted in a much higher C value indicating transcript detection was significantly less sensitive Random nonamers proved to be better than random hexamers or dodecamers data not shown the performance of random nonamers varies depending on the reverse transcriptase used gt Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Guidelines for successful reverse transcription This relationship between choice of RT primers and sensitivity of realtime RT PCR has been observed with many different transcripts and RT PCR systems Therefore when quantifying several targets from one RNA population we recommend using a mixture of oligo dT primers and random nonamers for reverse transcription The QuantiTect Reverse
102. scription ligation and amplification using Multiple Displacement Amplification MDA technology Figure 18 As reverse transcription takes place using a mix of random and oligo dT primers a cDNA library covering all transcript sequences including both 5 and 3 regions is prepared Figure 19 Subsequent ligation of the cDNA followed by MDA using a uniquely processive DNA polymerase generates amplified cDNA that preserves the transcript representation of the original RNA sample Figure 20 This is critical for accurate gene expression analysis Primer annealing Extension Displacement Multiple displacement SRS Figure 18 Schematic representation of whole transcriptome amplification cDNA is first synthesized from template RNA and then ligated not shown REPLI g DNA polymerase moves along the cDNA template strand displacing the complementary strand The displaced strand becomes a template for replication allowing high yields of cDNA to be generated EN D 20 o 15 2 sS gt CAP aaa 0 rsi 5 5 amplicon 3 amplicon 5 region 3 region Figure 19 Equal amplification of 5 and 3 regions EY Schematic diagram showing the location of the 2 amplicons with respect to the transcript J Total RNA 1 ng was amplified using the QuantiTect Whole Transcriptome Kit This was followed by real time PCR using 10 ng cDNA primers specific for B actin and the QuantiTect SYBR Green PCR Kit Amplicons corresponding
103. sult EY RNA DNA hybrids with high GC content e No amplification do not dissociate easily Primer binding and enzyme progression obstructed E RNase H removes RNA Yes Amplification in RNA DNA hybrids e Primer binds to single stranded cDNA and DNA polymerase proceeds In addition to Quontiscript Reverse Transcriptase the QuantiTect Reverse Transcription Kit also contains unique Quantiscript RT Buffer which is specifically designed to be compatible with downstream real time PCR analysis in contrast to common RT buffer formulations developed for other applications such as the preparation of cDNA libraries Quantiscript RT Buffer together with Quantiscript Reverse Transcriptase ensures accurate and sensitive results in real time two step RT PCR Figure 27 providing high yields of cDNA and helping to keep nonspecific products to a minimum When the QuontiTect Reverse Transcription Kit was used in real time two step RT PCR analysis of BCL2 transcript subsequent melting curve analysis showed a distinct peak in the melting curve for the specific product Figure 26 References 1 Omniscript and Sensiscript RT Kits for highly efficient reverse transcription QIAGEN News 1999 No 2 1 www qgiagen com literature qiagennews 2 Tacke E et al 1995 Transposon tagging of the maize Glossy2 locus with the transposable element En Spm Plant J 8 907 www qiagen com Real Time PCR Brochure 07 201
104. tStarTaq Plus DNA Polymerase enables highly sensitive and efficient one step RT PCR that is comparable to two step RT PCR Figure 29 A Two step RT PCR B One step RT PCR Amplification Plot 2 500 3 000 2 000 2 000 c 1 500 e e e lt lt 1 000 1 000 0 500 0 000 s rem 0 000 0 10 20 30 40 0 10 20 30 40 Cycle Cycle Figure 29 Comparable dynamic range and high sensitivity in two step and one step RT PCR Ubiquitin expression in human leukocytes was analyzed using a Primer Express designed TaqMan assay Reactions were run in duplicate on the ABI PRISM 7900 EY 10 fold cDNA dilutions 100 ng to 10 pg were analyzed using the QuantiFast Probe PCR Kit E 10 fold RNA dilutions 100 ng to 10 pg were analyzed using the QuantiFast Probe RT PCR Kit 8 5 Removal of genomic DNA contamination Contamination of RNA samples with trace amounts of genomic DNA can interfere with real time RT PCR quantification if the PCR primers used are also able to amplify genomic DNA sequences To avoid the negative effects of genomic DNA contamination careful primer design is required see section 9 7 page 52 If this is not possible RNA samples should be treated with DNase to digest contaminating DNA A faster alternative to DNase digestion is RNeasy Plus Kits which purify DNA free RNA using gDNA Eliminator columns For two step RT PCR applications the QuantiTect Reverse Transcription Kit provides cDNA synthesis with int
105. tection in realtime PCR 9 5 Realtime PCR with fast cycling 9 6 Real time RT PCR direct from cell lysates 9 7 Genomic DNA in realtime RT PCR 10 Guidelines for successful multiplex real time PCR 10 1 Guidelines for designing primers and probes 10 2 Guidelines for selecting appropriate reporter dyes and quenchers for the probes 10 3 Choice of PCR buffer 10 4 Guidelines for evaluating the performance of a multiplex assay 10 5 Guidelines for programming the real time cycler 10 6 Guidelines for analyzing data from a multiplex assay 11 Guidelines for successful virus load quantitation Selection Guide QIAGEN products for real time PCR and real time RT PCR Real Time PCR Brochure 07 2010 Sample amp Assay Technologies 31 31 33 33 3 3 37 37 42 43 45 46 49 52 54 54 54 535 57 58 58 61 62 Introduction 1 Introduction Realtime PCR and RT PCR are highly sensitive techniques enabling amplification and quantification of a specific nucleic acid sequence with detection of the PCR product in real time Quantification of DNA cDNA or RNA targets can be easily achieved by determining the cycle when the PCR product can first be detected This is in contrast with end point detection in conventional PCR which does not enable accurate quantification of nucleic acids Real time PCR is highly suited for a wide range of applications such as gene expression analysis determination of viral load detection of gene
106. the PCR buffer In addition care must be taken to design optimal primers and probes and to choose appropriate reporter dyes and quenchers If multiplex real time PCR under fast cycling conditions is desired shortening cycling times while maintaining reliable amplification of multiple targets is also a challenge 10 1 Guidelines for designing primers and probes E Try to keep the size of the amplicons as small as possible ideally 60 150 bp E Follow the guidelines for good assay design C Use specialized design software to design primers and probes L All assays should be designed using the same settings to ensure that they will work optimally under the same cycling conditions lB Check the specificity of your primers by performing a BLAST search www ncbi nlm nih gov blast Ensure that primer sequences are unique for your template sequence E For two step and one step RT PCR assays in which detection of genomic DNA must be avoided design primers or probes so that one half anneals to the 3 end of one exon and the other half to the 5 end of the adjacent exon see Figure 51 page 53 10 2 Guidelines for selecting appropriate reporter dyes and quenchers for the probes E For accurate detection of the different targets in a realtime multiplex PCR assay it is essential that the sequence specific probes are labeled with reporter dyes whose fluorescence spectra are well separated or exhibit only minimal overlap See Table 1 page 7
107. tically modified organisms GMOs SNP genotyping and allelic discrimination In this guide we provide information on the basic principles of realtime PCR terms used in real time PCR and factors influencing the performance of real time PCR assays We also cover the latest technologies in real time PCR including multiplex analysis fast cycling direct analysis of cell lysates and whole transcriptome amplification In addition we describe the advantages and disadvantages of different reaction chemistries as well as provide answers to frequently asked questions and guidelines for successful results Examples of the spectrum of research currently being carried out are also included We extend our thanks to those who have contributed to this project and hope that it may provide a useful guide to successful realtime PCR for researchers everywhere www qiagen com Real Time PCR Brochure 07 2010 Detection of PCR products in real time 2 Detection of PCR products in real time Real time PCR and RT PCR allow accurate quantification of starting amounts of DNA cDNA and RNA targets Fluorescence is measured during each cycle which greatly increases the dynamic range of the reaction since the amount of fluorescence is proportional to the amount of PCR product PCR products can be detected using either fluorescent dyes that bind to double stranded DNA or fluorescently labeled sequence specific probes 2 1 SYBR Green The fluorescent dye
108. tion in different cell amounts 10 cells 102 cells 10 cell 1 cel Amount of RNA ng 20 2 0 2 0 02 No of high copy transcripts 10 10 10 104 No of medium copy transcripts 10 104 10 10 No of low copy transcripts 10 10 10 1 No of mosaics transcripts 10 10 1 0 Complete representation of all transcripts t Stochastic problems for mosaic transcripts t Stochastic problems for low copy and mosaic transcripts Stochastic problems for low copy transcripts and loss of mosaic transcripts www qiagen com Real Time PCR Brochure 07 2010 Guidelines for successful reverse transcription 8 Guidelines for successful reverse transcription When performing real time RT PCR the primers and the enzyme for reverse transcription must be carefully chosen The primers should allow reverse transcription of all targets of interest and the reverse transcriptase should yield cDNA amounts that accurately represent the original RNA amounts to ensure accurate quantification In addition the effects of the components of the RT reaction on subsequent real time PCR must be minimized 8 1 Choice of RT primers The choice of primers for reverse transcription depends on whether one step or two step RT PCR is being carried out see section 3 1 page 9 In one step RT PCR the downstream PCR primer is also the primer for reverse transcription Therefore one step RT PCR is always performed with gene specific primers In
109. tly reverse transcribed and sensitively detected in real time PCR 1000 500 250 125 62 31 ngRNA Omniscript QIAGEN MMILV Supplier AMV Supplier P Real Time PCR Brochure 07 2010 Figure 28 Superior sensitivity and dynamic range Reverse transcription was carried out with different reverse transcriptases according to suppliers specifications using the indicated amounts of total RNA from Hela cells 1 20 of the reverse transcription reaction was used in a 25 cycle PCR amplification with QIAGEN Tag DNA Polymerase A 1 7 kb B actin fragment was amplified Sample amp Assay Technologies Guidelines for successful reverse transcription 8 4 Reverse transcription conditions for one step RT PCR The ideal reverse transcriptase for one step RT PCR should also exhibit the same properties as those described above for reverse transcriptases for two step RT PCR However one of the main problems in one step RT PCR is the inhibitory effect of the reverse transcriptase on the PCR step which can lead to increased C values and thus reduced sensitivity and specificity when compared with two step RT PCR QuantiTect and QuantiFast Kits for one step RT PCR have a patented additive in the reaction buffer to prevent this problem of PCR inhibition In addition other buffer components allow reverse transcription at high temperatures 50 60 C As a result the optimized ratio of reverse transcriptases to HotStarTag DNA Polymerase or Ho
110. to the 5 and 3 regions of the B actin transcript were detected with similar C values indicating that the QuantiTect Whole Transcriptome Kit provided equal amplification of all transcript regions www qiagen com Real Time PCR Brochure 07 2010 Guidelines for successful whole transcriptome amplification 7 1 7 4 A QIAGEN B Suplier R Targets not uniformly amplified 0 2 102 p d P 4 a a 10 gt 10 Relative transcript abundance WTA Relative transcript abundance WTA 1024 1054 1054 Z J J w z we 1074 gt 1074 Relative transcript abundance Control Relative transcript abundance Control Figure 20 Preservation of transcript profile E Total RNA 10 ng from Hela cells leukocytes or whole blood was amplified using the QuantiTect Whole Transcriptome Kit for 8 hours WTA As a control RNA was reverse transcribed without amplification using the Sensiscript RT Kit Control The expression of 15 different genes including NFKB IL8 CASP8 TP53 and GAPDH in the 3 samples Hela cells leukocytes and blood was then analyzed by real time PCR providing 45 independent analyses Relative transcript levels normalization to B actin from the WTA and Control samples were plotted against each other The blue dotted line indicates 100 correlation between the 2 data sets while the solid line indicates actual correlation As the data points are close to this dotted line this indicates that amplificat
111. trast to detection with SYBR Green primer dimers and other nonspecific PCR products do not contribute to the fluorescent signal when probes are used for detection However PCR results will still be affected by nonspecific amplification Nonspecific products can compromise reaction sensitivity and efficiency by competition between the desired PCR product and the nonspecific product This can reduce the dynamic range of the PCR and low template amounts and or genes expressed at a low level may not be detected The amplification efficiency see section 5 3 1 page 18 is lower and is often indicated by nonuniform C distances between dilutions of the template Improved PCR specificity leads to higher PCR efficiency sensitivity and dynamic range Figures 40 and 41 When expression levels of the human cytokine gene ILIRN were analyzed the QuantiFast Probe PCR Kit enabled sensitive detection from just 10 pg of cDNA template Figure 40A In contrast the kit from Supplier A allowed detection from 100 pg of cDNA template Figure 40B The increased sensitivity of the QuantiFast Probe PCR Kit is also reflected by its lower C values At the highest template amount 100 ng human ILIRN could be detected 2 cycles earlier with the QuantiFast Probe PCR Kit increasing the dynamic range of the reaction The C values obtained with the QuantiFast Probe PCR Kit were evenly spaced over the whole 10 fold dilution series Fluorescence norm SSRER
112. ul multiplex real time PCR 10 4 10 m Applied Biosystems 7500 Fast B Rotor Gene 6000 a TNF e INFG MYC HSP89 HSP89 FAM 285 rRNA Cy5 18S rRNA HEX TEP eee Template log Figure 55 Uncompromised sensitivity on any cycler 4 plex real time PCR was carried out using the QuantiFast Multiplex PCR R Kit and Primer Express designed TaqMan assays F Triplicate reactions run using 10 ng Ramos cell line CDNA EJ Duplicate reactions run using leukocyte cDNA 100 10 or 1 ng The graph of C values against CDNA amount shows that targets of greatly differing abundance were amplified with similar efficiency 4 plex data colored lines were comparable to singleplex data gray lines EX Singleplex analysis of t 8 14 and GAPDH Duplex analysis of t 8 14 and GAPDH 8 14 8 14 GAPDH e GAPDH 31 31 29 punem 29 poou 27 27 o 25 J 25 23 wwe 23 e 21 ee 21 19 4 T T T T 1 19 T T T T I 10 5 25 1 25 0 625 10 5 2 5 1 25 0 625 Template ng Template ng Figure 56 Linear C values over twofold decreases in template Duplex and singleplex PCR were carried out on the Applied Biosystems 7500 Fast System using the QuantiFast Multiplex PCR R Kit and assays for the t 8 14 chromosomal translocation and for GAPDH Quadruplicate reactions were run using genomic DNA from the Ramos cell line as template twofold dilutions from 10 ng to 0 625 ng When analyzing t
113. umber of standard RNA molecules can be calculated using the following formula X g pl RNA transcript length in nucleotides x 340 x 6 022 x 10 Y molecules pl g P Pi leng An alternative to the use of in vitro transcripts as RNA standards is the use of a defined RNA preparation e g from a cell line or virus preparation for which the absolute concentration of the target has already been determined 5 2 2 DNA standards for absolute quantification Plasmid DNA The most convenient way to create a DNA standard is to clone a PCR product info a standard vector Advantages of this method are that large amounts of standard can be produced its identity can be verified by sequencing and the DNA can easily be quantified by spectrophotometry Plasmid standards should be linearized upstream or downstream of the target sequence rather than using supercoiled plasmid for amplification This is because the amplification efficiency of a linearized plasmid often differs from that of the supercoiled conformation and more closely simulates the amplification efficiency of genomic DNA or cDNA After spectrophotometric determination of plasmid DNA concentration the copy number of standard DNA molecules can be calculated using the following formula X g pl DNA plasmid length in basepairs x 660 x 6 022 x 10 Y molecules ypl PCR fragment A PCR product containing the target sequence can also be used as a DNA standard We recommend includin
114. ut ROX dye see selection guide page 62 Kits supplied with a master mix containing ROX dye are intended for cyclers that require ROX dye for fluorescence normalization e g instruments from Applied Biosystems Kits supplied with a master mix free of ROX dye are recommended for all other cyclers as they allow greater multiplexing through the use of probes labeled with ROX Texas Red or other equivalent dye These include cyclers from Bio Rad MJ Research Cepheid Corbett QIAGEN Eppendorf Roche and Stratagene QuantiFast Multiplex Kits are also available in 2 formats see selection guide page 62 Kits supplied with a master mix containing ROX dye are optimized for use with all instruments from Applied Biosystems except the Applied Biosystems 7500 Kits supplied with a ROX free master mix and a separate solution of ROX dye are intended for use with the Applied Biosystems 7500 and cyclers from Bio Rad M Research Cepheid Corbett QIAGEN Eppendorf Roche and Stratagene The protocols provided with QuantiFast and QuantiTect Multiplex Kits have been developed on a wide range of realtime cyclers by QIAGEN Since all reaction and cycling parameters are already established there is no need to perform optimization steps such as determining limiting primer concentrations or adjusting cycling conditions Data demonstrating the performance of QuantiFast and QuantiTect Multiplex Kits in duplex triplex and 4 plex PCR can be viewed at www giagen com
115. y optimization steps With QuantiTect Multiplex Kits these steps are avoided multiplex reaction are amplified with the same efficiency as the PCR products in the corresponding single amplification reactions resulting in comparable sensitivities The master mix also provides high sensitivity and specificity with detection of as little as 10 copies of each target QuantiFast Multiplex Kits combine fastcycling real time PCR and RT PCR with the same advantages as QuantiTect Multiplex Kits A ready to use master mix containing QuantiFast Multiplex PCR Buffer and HotStarTaq Plus DNA Polymerase provides time savings of up to 50 in multiplex analysis on any realtime cycler and allows reliable quantification of up to 4 targets per reaction without the need for PCR optimization Reduced PCR run times are achieved through the use of HotStarlaq Plus DNA Polymerase and the PCR additive QBond see section 9 5 page 46 gt Real Time PCR Brochure 07 2010 Sample amp Assay Technologies Guidelines for successful multiplex real time PCR In addition to various salts and additives QuantiFast and QuantiTect Multiplex PCR Buffers contain a specially optimized combination of K and NH ions which promotes a high ratio of specific to nonspecific binding of primers and probes during each PCR annealing step see Figure 35 page 42 With these buffers primer and probe annealing is only marginally influenced by MgCl concentration so optimization b
116. y titration of Mg is usually not required The buffers also contain synthetic Factor MP which facilitates multiplex PCR by increasing the local concentration of primers and probes at the template and stabilizing specifically bound primers and probes Figure 53 m Nonspecific primer annealing B Specific primer annealing ner Primer Stobilizotion by Factor MP m mw Destobilizotion V lt of mismatched M a A primer by NH Template Template N Figure 53 Unique buffer promotes stable and efficient annealing EY NH ions prevent nonspecific primers and probes from annealing to the template E Synthetic Factor MP an innovative PCR additive increases the local concentration of primers and probes at the template Together with K and other cations synthetic Factor MP stabilizes specifically bound primers and probes allowing efficient primer extension by HotStarlaq DNA Polymerase or HotStarlaq Plus DNA Polymerase HotStarlaq DNA Polymerase and HotStarTaq Plus DNA Polymerase are inactive at room temperature and become active only upon incubation at 95 C This stringent hot start prevents the formation of misprimed products and primer dimers during reaction setup and the first denaturation step Competition for reactants by these PCR artifacts is therefore avoided see section 9 2 2 page 43 To suit the requirements of different real time cyclers QuantiTect Multiplex Kits are available with or witho

Critical Factors for Successful Real-Time PCR - Gene

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