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PrimeTime qPCR Application Guide

1. ee 84 8 2 3 AA 84 9 24 DECODED NEWSIEME s conos Nob ORR ER a nw IRE ded RE a 85 o Otel RESOUICES sorde dw oum Oo ACA SOROR XME E GY AA OE 85 Ook BLAS TAPIA SIS so y ros aa a aa a Ea 85 8 3 2 RI qPCR Data Analysis a 85 9 References O III ww eee qx 86 10 Notice of Limited Licenses o 88 AXCXIDI INTEGRATED DNA TECHNOLOGIES 1 Introduction This gPCR Application Guide is intended to provide guidance on the entire qPCR process from RNA isolation to data analysis This document should be used to obtain a basic un derstanding of everything involved in the experimental setup performance and analy sis The guide begins with a general overview of qPCR and then provides more specific information on design experimental setup data analysis and troubleshooting for the 5 nuclease assay A general protocol for using PrimeTime qPCR Assays for this process is included Importantly this document follows the recommendations provided in The MIQE guidelines Minimum Information for Publication of Quantitative Real Time PCR Experi ments and two recent updates 1 which are a definitive guide and excellent resource for all of the necessary requirements for experimental setup analysis and publication 1 1 Advantages of qPCR Quantitative real time PCR qPCR has become the most precise and accurate method for analyzing gene expression Prior to qPCR the most common methods for determin in
2. 1 000 E 1 1 000 E 1 1 000 1 000 ARn ARn ARn 1 000 E 2 1 000 E2 1 000 E2 2 2 Cycle 10 15 30 5 Detector F AM1 E Plot ARn vs Cycle y Thresholct 0 06114806 0 2 30 15 2 2 Cycle Cycle 0 5 10 15 30 5 Detector F AM1 E Plot ARn vs Cycle Y Threshold 0 09851233 Detector F AM1 E Plot ARn vs Cycle y Threshold 0 057102246 A 60 Sec Anneal Extend B 45 Sec Anneal Extend C 15 Sec Anneal Extend Figure 19 Assay Results When Run at Different Cycling Conditions Reactions were run in 384 well plates using the Applied Biosystems Gene Expression Master Mix 10 uL per reaction and a 5 log dilution of Human Universal Reference RNA cDNA When the anneal extend step becomes too short C amplification is inconsis tent and decreased or even prevented Annealing temperature An annealing temperature that is too high can result in no am plification while one that is too low can result in nonspecific amplification or amplifica tion of primer dimers Change the annealing temperature in 2 C increments Cycle number Too few reaction cycles can result in little or no amplification The maximum number of cycles varies from 35 to 45 but the ideal number of cycles is 40 Most researchers disregard Cg values greater than 38 because amplification beyond cycle 38 is indicative of inefficient PCR 65 aPCR Application Guide 7 1 3 Primer or Probe Integrity Primer problems Determine whether reduce
3. 20 C protected from light See Section 4 3 1 for a resuspension protocol Probe degrading while cycling Run a reaction with the probe alone no primers to see if the signal increases If there is a significant increase in fluorescence it is likely that the probe is being degraded during cycling due to the presence of contaminating nucle ases Also be sure to run a no template control containing probe and primers alone this should show low flat line fluorescence or slightly increasing fluorescence Follow good laboratory practices to avoid introducing nuclease contamination Poor quenching High background can also be caused by poor quenching of the probe Probes longer than 30 bases with a single quencher at one end may have poor quench ing ability Design probes to be shorter than 30 bases unless using double quenched probes see below If there is a long run of As or Ts the addition of LNA bases can help raise the Tm Poor quenching may also occur ifan inappropriate quencher is being used for the assay Verify that the quencher and fluorophore are a good pair See Section 4 2 2b for proper design of probes and Table 3 for absorbance ranges of quenchers and fluorophores IDT offers dual quenched probes that contain a ZEN quencher internally in addition to a quencher on the 3 end of the probe Such dual quenched probes can resolve problems associated with poor quenching by significantly decreasing background and increas ing signal inte
4. XIDT INTEGRATED DNA TECHNOLOGIES Amplification Plot Slope 3 2692 R 0 9994 Log Quantity ie Detector All Plot ARn vs Cycle y Threshold Figure 16 Range of Dilution Over Several Logs A 6 point 10X dilution series over 5 orders of magnitude 1 x10 to 1 x 10 copies was created for the JAK2 transcript Amplification was performed using Gene Expres sion Master Mix Applied Biosystems on the Applied Biosystems 7900 Real Time PCR instrument under standard cycling conditions 2 min 50 C 10 min 95 C 45 x 15 sec 95 C 1 min 60 C A The resulting amplification curves show a steady decrease in the threshold cycle with increasing sample concentration For a reaction that is 10096 efficient the threshold cycle should increase by 3 3 cycles for each 10X dilution i e it takes 3 3 cycles for the product to amplify10X B Linear regression of the amplification curve data derived from the dilution series gen erates a standard curve For a reaction that is 100 efficient the slope of the line should be 3 3 for a standard curve generated from a 10X dilution series When absolute standards are used e g synthetic templates long oligonucleotides the y intercept represents the Cq value for a single molecule 5 3 PCR Efficiency The efficiency of qPCR is influenced by many factors including target length target se quence primer sequence buffer conditions impurit
5. 17 The baseline END value should be at a Cg value two cycles before the amplification curve for the highest expressing sample crosses the threshold Setting the baseline is instrument dependent For more information on setting the baseline please refer to the user manual for the instrument you are using 52 XIDT INTEGRATED DNA TECHNOLOGIES Linear View Log View Amplification Plot Amplification Plot Amplification Plot Amplification Plot 10 15 20 25 Cycle Detector FAM Plot ARn vs Cycle Y Threshold 0 20 20 25 Cycle Detector F4M Plot ARn vs Cycle Y Threshold 0 20 Panel A Linear Plot Panel B Log Plot Figure 17 Plotting Amplification Data 6 3 Setting the Threshold The threshold is the ARn level that determines the threshold cycle or Cg The threshold is set on the baseline corrected from relative fluorescence In Figure 18 it is seen in log view as a line above the baseline and through the amplification curve in the exponen tial growth region linear increasing signal in a log scale The Cg is the cycle number at which the amplification plot crosses the threshold 3 Setting the threshold is instru ment dependent For more information refer to the user manual for the instrument you are using Amplification Plot 1 000 Figure 18 Proper Threshold Setup The threshold is indicated by a green line through the curve in the exponential regi
6. The Agilent 2100 Bioanalyzer Agilent Technologies or the Experion system Bio Rad Labo ratories can be used for this purpose These instruments enable electrophoretic sepa ration of very small amounts of RNA sample which can be detected by laser induced fluorescence High quality eukaryotic RNA will have both 185 and 28S rRNA peaks with the 285 region in greater abundance and a low amount of 55 RNA 14 Figure 7 The RNA integrity value is determined from the shape of the resulting electropherogram curve and is based on several characteristics The software uses an algorithm to assign a number to the RNA with 1 being the most degraded and 10 being the most intact 14 The ideal integrity value will depend on the RNA source as some tissues will provide higher quality RNA See the publication from Fleige and Pfaffl 14 for more information on average integrity values for various tissues RNA Quality Analysis Figure 7 Examples of Sample Qual ity Experion BioRad microfluidics analysis of poor quality RNA Sample 1 and good quality RNA Sample 2 The 28S rRNA species is more sus ceptible to degradation Sample 1 Tenn 8 3 8 8 Ss S m S s 8 s o o 2 s 8 6 s Sample 2 o it 18S rRNA 28S rRNA Y N aPCR Application Guide The ratio of 260 280 nm absorbance readings can also give an indication of RNA quality However other contaminants may affect this ratio making it less accurate tha
7. ios deae ced dk des xe x deo dee he ede Pega e abe e e 55 6 4 2b Efficiency Corrected Gene Expression Measurements o 56 6 5 Qualitative JATISIVSIS ss krank oO de aia oe ek deg qx c A e C d 58 7 PrimeTime qPCR Assay Troubleshooting 59 7 1 Little to No Amplification e 64 Lul REICO c c 64 NA eeu o 34 4 od u 4 6s 28 owe oe ee O ee Oe Se See 64 Z3 Primer Or Prope IOtegrlty uas sy ak om bc aE Re OG de RR REGO on 66 7 1 4 Sample Expression 66 72 Low or Delayed IO sace dcr de eR RE Y ede RAB EER ECCE nde d des 67 7 24 DEON SpeCIHCID ooo cae se Soe be a wA X Geo ODE Ge UR RAS 67 250 2 ss aa DU MAI 67 7 2 3 Sample Expression 68 J 2A DOSING amp ax dm aac ana A Oe AS MAES Ue BERD DD HO 68 JISENOICCO DYES oso raras dr Phe ee ee ede naw e X UE s 69 7 2 POOF ECEN corea henee Oe abre a 70 AXCXIDI INTEGRATED DNA TECHNOLOGIES 434 DESIGN Speclielb uuu x wer orem RE GUREOE RPESTXRIEYSSG Ed wes x XR S 70 oL RECO OCU Die a a ay Ga aed eo d ge sri 3E aede enc ag EC a ana 70 123 MSM Le seas 0 do a adas a AAA 70 7 4 Excessive or Unexpected Signal ee Z1 7 4 1 Instrument Calibration 71 TAL Assay SPECINCIY acd xine oe bah ntaa e REOR SOR cn ee Ree ee eed RES 71 JAD COMMON uus sra bow Se dou e Cede a eh ze p ee oe 71 7 4 4 Template Concentration eaea a es 12 E g c p T CC TTITTT 72 7 6 Inconsiste
8. 50 ng cDNA with FAM or HEX dye Cq values were 22 8 for FAM singleplex ARn Singleplex iinet 22 5 for FAM multiplex and 25 for HEX singleplex and multiplex HEX Multiplex 1 000 E 1 f it LETT ae TTT 0 5 10 15 20 25 30 35 40 45 Cycle Detector All Plot ARn vs Cycle Thresholet 35 aPCR Application Guide 4 2 5 Calibrating Dyes for Multiplex gPCR Multiplex qPCR allows researchers to perform multiple assays simultaneously in a sin gle well The advantages of this technique include increased efficiency in sample and resource usage higher throughput and faster time to results and elimination of well to well variations In multiplex qPCR fluorescent dyes with distinct spectra are used to detect individual targets Thermal cycler and real time detection system emission and excitation filter settings vary from manufacturer to manufacturer so the instrument must be calibrated for each dye as part of the experiment optimization process to en hance dye specificity and minimize background and overlapping of fluorescent signals General Calibration Protocol Calibration procedures are specific to each real time PCR instrument In general most calibration protocols require the following steps Perform background calibration procedure Dilute dye in amount of reaction buffer recommended by the manufacturer Aliquot amount of recommended dilution into a PCR plate and seal use different plates fo
9. A Amplification Curves for PrimeTime qPCR Assay 0 vs 30 Freeze Thaws Rn vs Cycle Amplification Plot Amplification Plot Detector FAM1 Plot Rn ws Cycle y Panel B PrimeTime qPCR Assay Normalized View vs Cycle Figure 8 PrimeTime qPCR Assays are Stable After 30 Freeze Thaw Cycles A A standard scale PrimeTime qPCR Assay was hydrated in IDTE to 40X The tube was frozen 20 C and thawed 30 times At 0 15 and 30 freeze thaws an aliquot of the assay was run against a validated uni versal human reference cDNA standard curve 0 005 50 ng using Taq Man Gene Expression Master Mix Applied Biosystems B The Prime Time qPCR Assays at 0 5 ng cDNA concentration in a ROX normalized view Rn PrimeTime qPCR Assays showed no probe degradation and no impact on C value up to 30 freeze thaw cycles 20 AXCXIDI INTEGRATED DNA TECHNOLOGIES 3 Perform Reverse Transcription Transcription is the synthesis of RNA from a DNA template reverse transcription RT is the synthesis of DNA from an RNA template DNA synthesized from RNA is often referred to as first strand cDNA The conversion from RNA to cDNA is necessary because PCR uses DNA dependent polymerases The exact reaction conditions are dependent upon the particular kit or protocol used but all contain the same basic components the RNA to be converted dNTPs to provide the nucleotides for cDNA synthesis primers buffer DTT to stabilize t
10. The expression level of the reference gene should be the same across all conditions See Section 6 4 2a for more information on normalization Hs PGK1 Hs SFRS9 Amplification Plot Amplification Plot 1 000 E 1 1 000 E 1 j 1 0004 1 000 E 1 4 1 000 E 2 f 1 000 E3 Detector FAM 1 Plot arn vs Cycle y Threshold 0 11410569 Detector FAM 1 Plot arn vs Cycle y Threshold 0 11410569 A Hs PGK1 B Hs SFRS9 Figure 24 Choosing the Most Stable Normalizer RNA was isolated and cDNA generated from 18 wells of HepG2 cells transfected with control DsiRNA Assays targeting PGK1 or SFRS9 were performed The SFRS9 assay curves showed a smaller degree of variance compared to the PGK1 assay curves 81 aPCR Application Guide 8 RI qPCR Additional Resources 8 1 MIQE Publications The information in this guide is designed to follow the standards outlined in the MIQE Guidelines Minimum Information for Publication of Quantitative Real Time PCR Ex periments 1 The purpose of the MIQE guidelines is to provide a minimum set of re quirements for conducting and analyzing RT qPCR experiments and to enable other researchers to replicate findings It is recommended that investigators using RT gPCR for their own experiments review those guidelines together with this guide as it will aid in assay performance reproducibility and publication of experimental data Two updated publications M QE precis Practical implementation o
11. These events can be de tected by combining melt curve and size analysis or performing the more detailed se quencing analysis 5 1 3 Sequencing Sequencing of PCR products especially using massively parallel deep sequencing pro vides the most thorough validation of specificity but at significant cost and effort 46 AXCXIDI INTEGRATED DNA TECHNOLOGIES 5 2 Efficiency Analysis 5 2 1 Standard Curves There are 2 types of standard curves absolute or relative standard curves An absolute standard curve is created by diluting a nucleic acid sample typically a plasmid oligo nucleotide or purified PCR product that has been accurately quantified by other means A dilution series of this sample is prepared and each dilution serves as a standard To ob tain the most accurate quantification the amplification efficiency of the standards must be equivalent to that of the test samples The standards are assayed simultaneously with the test samples and a standard curve is generated from the dilutions The concentration or number of copies of the test samples can then be determined through interpolation from the standard curve Creating a standard curve requires setting up additional reac tions however the data obtained can be very important for determining the quality of the PCR Relative standard curves are generated by serially diluting a sample whose target con centration is not known e g cDNA prepared from a total RNA sample fo
12. amp B 10 nM is too low ARn will be high and noisy and E amp F 100 nM is too high ARn will be low 9 aPCR Application Guide 7 9 Multiplexing Problems Inability to detect expression of a target in the multiplex reaction that could be de tected when analyzed individually Increasing the number of genes to be analyzed in a single qPCR requires increasing the concentration of several of the PCR components including MgCl and dNTPs Using a master mix specifically designed for multiplex PCR is recommended Make sure all of the primers and probes have similar melting temperatures The melting temperatures of the probes should be 6 10 C higher than those of the primers Also evaluate the cross reactivity of each assay component to ensure there is no interac tion between primers and probes The OligoAnalyzer Tool is ideal for this purpose See Section 8 2 2 for more information For more information on setting up a multiplex assay see Section 4 2 4 Cq values for the targets in the multiplex reaction look different from those for the targets when they are analyzed separately The Cy values should be similar whether a target is tested in a single reaction or a multiplex reaction Limit the primers for the highest expressing targets to a 1 to 1 primer to probe ratio Use double the amounts of dNTPs and enzyme in the master mix The Mg concentration may also need to be adjusted See Section 4 2 4 for more information on setting up a
13. analysis tools that include the PrimeTime gPCR Assay Design Tool for identifying current Prime Time Predesigned qPCR Assays the RealTime PCR and PrimerQuest design tools for designing primers probes and assays OligoAnalyzer program for analyzing oligonucleotide melting temperature hairpins dimers and mismatches and UN AFold program for analysis of oligonucleotide secondary structure For more information to order any of these products or to use the free design tools visit the IDT website at www idtdna com AXCXIDI INTEGRATED DNA TECHNOLOGIES 1 3 qPCR Workflow The typical qPCR experiment involves the following steps Sample collection RNA isolation and quality control Section 2 Reverse transcription Section 3 Real time PCR Section 4 Assay validation and data analysis Sections 5 and 6 Collect Isolate Sample RNA Perform Reverse Perform Validate and Transcription Reaction qPCR Analyze qPCR Each of these steps is covered in this guide along with recommendations for proper ex perimental setup and design All of the recommendations follow the MIQE guidelines See Section 8 1 and reference 1 aPCR Application Guide 2 RNA Isolation and Quality Control The first steps in running a qPCR assay are to collect the sample and isolate total RNA The method of RNA isolation will depend on the sample type and experimental conditions After isolation both the quantity and quality of the RNA shoul
14. and CODITOIS S a sug s oRacRo Shee D EO OR ER RU aL basen e x Pob wm 22 a S once ou bee sed v 5e Pe ew cH Ede wx SV oe en d 27 3 2 Dnesstep vs TWO SEPD RIS e uc o aa a o oae OR ds e da o os A 22 3 6 Example Reverse Transcription Reaction Protocol o 23 4 Real Time qPCR Design and Protocols 24 4 1 PrimeTime qPCR Assays and Associated Products o o 24 41 1 Primelime Custom GPCR ASSAYS ss ww Ghee we dos ESE Ome Eee Re 25 4 1 2 PrimeTime Predesigned qPCR Assays o ee en 25 4 1 3 ZEN Double Quenched Probes e 25 4 2 5 Nuclease Assay Design ee 27 4 2 1 General Design Considerations oaaae 27 2 7 2 Primer ANG PODES uu arat aa as 3 2 9p mos Y p a ee Bae 28 ALLA PMES xs que aceto perra dod KOREA SOA 28 AIO PODOS a oak Ad A ARA A aa A 29 AZLCIAMPICIAS rana rara A E 30 4 2 2d Calculating Melting Temperature Tm 30 4 2 3 Choosing the Correct Reporter Dye for the Instrument 32 ADAM PIE O PER ses kee ee beep beh oe been ESRB Owe a A 34 4 2 5 Calibrating Dyes for Multiplex qPCR 2 2 ee 36 426 Replicates a a CONTOS ess aks pero a ea bd de ox eee Ey Ree a Yoges 36 4 2 6aReplicates 2 ee 36 42 60 Negative Controls es Knee DR OR ERED MARY CERO y eX wR ox 38 aPCR Application Guide gPCR Application Guide Experimental Overview Protocol Troubleshooting 42 00 Positive COMUO S 24 oun
15. by the dye During PCR the primer binds to the target for the first round of target synthesis Because the primer and probe are connected the probe becomes attached to the newly synthesized target region The spacer region prevents the DNA polymerase from replicating the probe sequence disrupting the stem structure and rendering the amplicon permanently fluorescent e When the second cycle begins binding of the loop sequence to the amplicon is thermodynamically favored over binding to the hairpin stem The probe is dena tured and hybridizes to the target separating the fluorophore and quencher The resulting fluorescence emission can be detected during the annealing phase 1 2 5 Intercalating Dyes AXCXIDI INTEGRATED DNA TECHNOLOGIES Intercalating dyes are nonsequence specific fluorescent dyes that exhibit a large in crease in fluorescence emission when they intercalate into double stranded DNA Ex amples include SYBR Green I Cyto EvaGreen and LC dyes 10 11 During PCR the primers amplify the target sequence and multiple molecules of the dye are inserted between bases of the double stranded product causing fluorescence Figure 6 Figure 6 Intercalating Dye During the annealing step the primers hybridize in a sequence dependent man ner to the complementary DNA strand In the extension step the polymerase begins DNA synthesis extending from the 3 ends of the primers As the amplicon is ex tended the intercalating
16. control to identify erroneous signal due to genomic DNA contamination This reaction has all of the components of the other reactions but the reverse transcriptase is left out This control will be very useful later in the qPCR step as a negative control 3 4 CDNA Storage Aliquot the cDNA samples and store the first strand cDNA at 20 C 3 5 One Step vs Two Step RI gPCR There are commercially available products for performing the RT reaction and gPCR in a single step one step qPCR This may be a good option if you plan to use the cDNA for only a limited number of assays However if you are interested in making a large amount of cDNA to use for multiple assays two step qPCR is recommended In addition one step qPCR can be less sensitive than two step and prevents you from varying the amount of input cDNA For more information on this subject see the article Starting with RNA One Step or Two Step RT qPCR in the IDT DECODED 1 3 October 2011 news letter at www idtdna com 22 AXCXIDI INTEGRATED DNA TECHNOLOGIES 3 6 Example Reverse Transcription heaction Protocol 20 uL reaction volume for two step RT qPCR 1 Combine the following components 1 uL 2 uM gene specific RT primer or 250 ng oligo dT 1 uL dNTP mix 10 mM each 10 5 uL total RNA 10 ng uL 100 ng 2 Heat at 65 C for 5 min and then chill on ice 3 Add 4 uL 5X First Strand Buffer 2 uL 0 1 M DIT 1 uL RNase inhibitor such as RNasin Ribonuclease Inhibito
17. dye binds to the newly formed double stranded DNA Fluorescence increases in a sequence independent manner with increasing ampli con length When DNA synthesis is completed the final amount of fluorescence a function of both the length and number of copies of the amplicon is determined Finally during denaturation the dyes dissociate from the amplicon Intercalating fluorescent dyes are not specific to a particular sequence thus they are both inexpensive and versatile As they can bind to any double stranded sequence they will also bind to primer dimer artifacts or incorrect amplification products 12 Therefore when using interca lating dyes itis important to analyze the melting curve of the amplicon to ensure that the primers are amplifying a single product observed as a single melting curve peak These types of dyes cannot be used for multiplex analyses as the different PCR prod ucts would be indistinguishable See Section 4 2 4 for more information on multi plex reactions Because multiple dye molecules intercalate into a double stranded product the in tensity of the fluorescent signal is dependent on the mass of the amplified product Assuming both amplify with the same efficiency a longer product will generate more signal than a shorter product 8 In contrast probes are specific to a particular sequence and will emit the same amount of energy from a single fluorophore irre spective of the length of the amplified p
18. few months For longer term storage freeze and store the RNA at 80 C or precipitate the RNA and store it in ethanol at 20 C 2 2 Quantify RNA can be quantified by several methods including UV spectrophotometry microflu idic analysis capillary gel electrophoresis or by the use of fluorescently labeled RNA binding dyes 1 Absorbance measurements at 260 nm on standard spectrophotome ters can be used for quantification when RNA is abundant while the NanoDrop Thermo Scientific and DropSense Trinean instruments are useful for measuring limited quanti ties of sample Microfluidic quantification can be performed using the 2100 Bioanalyzer Agilent Technologies or the Experion system Bio Rad Laboratories These microfluidic AXCXIDI INTEGRATED DNA TECHNOLOGIES methods also enable RNA quantification of limited amounts of sample In addition mi crofluidic analysis enables simultaneous integrity assessment see Section 2 3 below 2 3 Check Quality In addition to having similar quantities of RNA it is also important that the samples be of similar quality The quality of the RNA can have a large impact on the results of the ex periment poor quality RNA can compromise the entire experiment and result in wasted time and money Furthermore differences in quality between two samples can lead to misinterpretation of gene expression differences RNA quality can be assessed most accurately by calculating the integrity of the RNA
19. for 10 sec prior to opening in case any material was dislodged during shipment Resuspend the assay in IDTE buffer 10 mM Tris 0 1mM EDTA pH 8 0 at the volumes indicated in Table 5 a Resuspend PrimeTime qPCR Assays as 40X 20X or 10X stocks A particular con centration may be preferred depending on the size of the assay and desired final reaction volume see Table 5 below b Vortex the sample to ensure maximal product recovery Centrifuge the resuspended assay at 750 x g for 10 sec The resuspended assays will yield a final 1X concentration of 500 nM primers and 250 nM probe when ordered using default conditions Standard or XL Assays ordered using a custom primer to probe ratio other than 2 1 will yield a different concentration of primers 40 AXCXIDI INTEGRATED DNA TECHNOLOGIES Recommended Resuspension Volumes for PrimeTime Assay Stock Creation Final Desired Stock Concentration 40X 20X 10X PrimeTime Mini qPCR Assay Not recommended 100 uL 200 uL PrimeTime Std qPCR Assay 250 UL 500 uL 1 000 uL PrimeTime XL qPCR Assay 1 250 uL Not recommended Not recommended Table 5 Recommended Resuspension Volumes for Producing PrimeTime qPCR Assay Stock Solutions helated Products at IDT IDTE buffer IDT offers 1X TE Buffer 10 mM Tris pH 7 5 or 8 0 0 1 mM EDTA for initial resuspension and storage of DNA oligonucleotides DNA oligonucleotides can be damaged by prolonged incubation or storage in even mildly acidic
20. quencher no longer absorbs the fluorescence emitted by the dye This fluorescence is detected by the real time PCR instrument Meanwhile the polymerase continues extension of the primers to finish synthesis of the DNA strand aPCR Application Guide 1 2 2 Molecular Beacons e Molecular beacons are labeled with a 5 fluorescent reporter dye D and a 3 quench er Q A stem region is designed such that at annealing temperature and in the absence of target the ends of the beacon are held closely together which allows the quencher to absorb the fluorescence from the reporter Figure 3 When the probe hybridizes to its target sequence during the annealing phase of the PCR the quencher is no longer in close proximity to the fluorophore dye and the reporter fluoresces 8 The resulting fluorescence is measured during this annealing phase he uncleaved probe dissociates during the extension step and can participate in the next PCR cycle e Molecular beacons will thermodynamically favor the hairpin structure over a non specific target sequence which makes these probes highly specific A perfect match probe target hybrid will be energetically more stable than the stem loop structure whereas a mismatched probe target hybrid will be energetically less stable than the stem loop structure 9 IDT synthesizes the probes and primers needed for this system TE o Figure 3 Molecular Beacons During the annealing step the prim
21. temperatures These modified enzymes require activation by heating at 95 C for 2 10 minutes during the initial denaturation step ROX dye some thermal cyclers require the use of an internal reference dye such as ROX dye for normalization across wells and to account for pipetting errors There fore certain master mixes are available with different formulations of ROX Refer to your instrument user manual for instructions Related Products at IDT PrimeTime Gene Expression Master Mix This 2X master mix antibody mediated hot start DNA polymerase dNTPs MgCl2 enhancers and stabilizers is optimized to support 2 step qPCR probe based assays for gene expression analysis It is compatible with standard or fast cycling conditions and overnight experiments Learn more at www idtdna com qPCRmastermix 44 HPRT FAM labeled probe Singleplex o 3 c S 20 E y O 15 10 1 0 00 02 0 5 0 40 35 GUSB HEX labeled probe Singleplex 30 N o 2 20 y c o ce 15 lt 21 26 Cycle 1 0E 01 1 0E 02 1 0E 03 1 0E 04 XIDT INTEGRATED DNA TECHNOLOGIES Singleplex Duplex FAM dye Efficiency 97 53 Efficiency 95 58 R 0 9999 R 0 9998 1 0E 03 1 0E 04 1 0E 05 10E 06 1 0E 07 1 0 08 of copies Singleplex O Duplex Singleplex Efficiency 96 71 R 0 9999 Duplex HEX dye Efficiency 98 05 R 0 9999 10E 05 10E 06 10E 07 10E 08 of copies o Singleplex a D
22. value attributed to ROX and consequently the Cg values reported Resolve this by using another dye with an emission spectrum that does not overlap the ROX absor bance range 78 10 nM ROX too low Panel A Amplification Curves at Low ROX Concentration 50 nM ROX correct concentration Cycle c Threshole 0 51572704 Detector fam 7 Plot farn vs Cycle Panel C Amplification Curves at Correct ROX Concentration 100 nM ROX too high Cycle yde Thresholet 0 09977144 Panel E Amplification Curves at High ROX Concentration XIDT INTEGRATED DNA TECHNOLOGIES Multicomponent Welk B3 Background Fluorescence Time hh mm Panel B Multicomponent Plot of Assay Dyes Multicomponent Welk AS Multicomponent Plot Fluorescence Time hhmm Panel D Multicomponent Plot of Assay Dyes Multicomponent Well C3 Fluorescence Time hh mm Panel F Multicomponent Plot of Assay Dyes Figure 23 Using the Correct ROX Concentration ROX concentrations were tested using Immolase DNA Polymerase Bioline 250 nM Probe 500 nM primers Assay NM 004530 2 0 8 mM dNTP and 3 mM MgCl 10X serial dilutions of cDNA from Human Universal Reference RNA 20 0 002 ng were amplified 10 min 95 C 45 x 15 sec 95 C 1 min 60 C C D 50 nM ROX is the optimum concentration for this assay while A
23. which will provide a known amount of product can be purchased from IDT Cloned am plicons Ultramer Oligonucleotides and gBlocks Gene Fragments provide ample prod uct to create a 7 log standard curve As a third option a dilution series of cDNA can be prepared by reverse transcribing RNA from a tissue or cell line that expresses high levels of the target of interest Pooled RNA from multiple cell lines is also commercially avail able It is important to get as many data points as possible with at least 4 7 points for the Standard curve to obtain a reliable estimate of the reaction efficiency Related Products at IDT Ultramer Oligonucleotides and gBlocks Gene Fragments serve as excellent con trols and importantly can be used as standards of known concentration For more information and to order Ultramer Oligonucleotides and gBlocks Gene Fragments visit the IDT website at www idtdna com Ultramer Oligonucleotides IDT synthesis systems and chemistries allow high fideli ty synthesis of very long oligonucleotides up to 200 bases Suitable for demanding applications such as cloning ddRNAi and gene construction Ultramer Oligonucle otides can save researchers a lot of time and effort because the entire fragment is directly synthesized by IDT gBlocks Gene Fragments IDT offers double stranded DNA fragments up to 500 bp in size gBlocks fragments are constructed using Ultramer Oligonucleotides and are sequence verified 48
24. 4 5 See Section 4 2 4 for more information on multiplex reactions When the primers and probe hybridize to the target and the polymerase begins to extend the primers the probe is hydrolyzed by the 5 to 3 exonuclease activity of the polymerase causing the reporter and quencher s to dissociate from the target The spatial separation of reporter and quencher s disrupts the ability of the quencher s to absorb energy emitted from the reporter and thus a substantial increase in re AXCXIDI INTEGRATED DNA TECHNOLOGIES porter dye fluorescence occurs 6 The fluorescence produced during each cycle is measured during the extension phase of the PCR Examples of 5 nuclease assays include PrimeTime qPCR Assays IDT and TaqMan Assays Applied Biosystems See Section 4 1 for more information about PrimeTime qPCR Assays and ZEN double quenched probes Figure 2 5 Nuclease Assay During the annealing step the primers and probe hybridize to the complementary DNA strand in a sequence dependent manner Because the probe is intact the fluorescent reporter D and quencher Q are in close proximity and the quencher absorbs fluorescence emitted In the extension step the polymerase begins DNA synthesis extending from the 3 ends of the primers When the polymerase reaches the probe the exonuclease activity of the polymerase cleaves the hybridized probe As a result of cleavage the fluorescent dye is separated from the quencher and the
25. 7900HT o O e X e Rx x X x Applied Biosystems QuantStudio 6 7 e e Rio O Applied Biosystems QuantStudio 12K e fe o o O e R IO O Ol O Applied Biosystems StepOne 0 0 X X Rx x x x Bio Rad CFX384 CFX96 lolelo o o o lo elo l Bio Rad MiniOpticon MiniOpticon II MylQ2 Bio Rad iQ 5 e O s O O a O O O a Cepheid Smartcycler Smartcycler II e O O X olo e O A Eppendorf Mastercycler e e e Qiagen Rotor Gene Q Rotor Gene 6000 o o o e x X o e X Roche LightCycler 480 e O o O O O0 O e Roche LightCycler 1536 e O O O O O O JO O OL O Roche LightCycler Nano O olololo e O Agilent Mx3000P Mx3005P e e Dye compatibility of the Mx3000P or Mx3005P instruments depend on the filter selections made during purchase Table 2 Instrument Supplier provided or recommended Compatibility with Reporter Dyes O Instrument supported dyes that may require calibration R Instrument uses channel for the reference dye X Not an instrument supported dye 33 aPCR Application Guide 4 2 4 Multiplex qPCR In multiplex PCR multiple targets are amplified in a single reaction tube Each target is amplified by a different set of primers and a uniquely labeled probe that will distin guish each PCR amplicon Multiplexing provides some advantage
26. PCR Application Guide Experimental Overview Protocol Troubleshooting Fourth Edition 2015 Integrated DNA Technologies Inc All rights reserved This material may not be reproduced in whole or in part without the express prior written permission of the copyright holder Permission granted to reproduce for personal and educational use only Commercial copying hiring lending is prohibited AXCXIDI INTEGRATED DNA TECHNOLOGIES qPCR Application Guide Experimental Overview Protocol Troubleshooting Im indes arriero nara AREA 7 LT Advantages ol gPCRs aunar dede aa Bero 3e 7 1 2 1 5 Nuclease ASSAY qo x we eo HY RT oom gem Fe ERED da GH s 8 192 2 Molecular BedOOFIBa s a ae ve de ad ce de AOR A doc es db o HOR ae OL I I 10 1 2 3 HybrigizatiornVFRET PIODES uu 0444455444440 PX eee a een ae das 11 L24 SCOIDIONG PODES os x eux wound RES S xSsm4 ae wee oe eS 12 12 5 iia o an a ao auem GAY eoe e SOR MEG dora SEE HHH HOS 13 Lo POR IVONNE ko 2a an phe eden ig ow RS aa Bad 15 2 RNA Isolation and Quality Control lll 16 A oi asp 4 Roars 4 ee ew 4 ee od GG A RO ge SS wh ep ew ea 16 2 2 CUBE sers qose bo E ar A 16 Ege ALO e 2 gi os Gow oy eo ee ee Re ee Ee eR Od ee ee 17 24 volg RNases DINaSBSu s ue eek we RAR a a E Ce Re OE Oy des 18 3 Perform Reverse Transcription 21 o oa he ea ew wae wae ee ee Maes ae eee eee ee ee Ped 21 32 Choice of Primers aaa rs 21 3 3 Repllcates
27. Primelime OU Ik Jet OUXUL Orva JEUX RUFO J uU uU XIVUUVUU r rLrur UU UUU C UUUUUULI FRUFFUUXXF MUERE IIIPLC JUDVDFUUVUUOXU JVUVUVVUVUUFUX lt XIVUXIVUUFUUE IRE UVFRFUOUFU REXFFPOUOPUIU JUDERFRUIIVURE YIUXIUVUFFUU TIVUUVUFRUXIF U lt YIIVIUVUFEIOXU VUUFUUDUUDORU VUUXIIFUUUUX UUcuUrauruu 2 ur ur VUFUFRUFUFU UUDUUFUXFUF IVEHIIIUXO IVUFUEHEHXIIX J rrLr uc DOrFCUUUC FUUUFUU Q rr r ur XIUUUVUUO LLULILL IIVUFO VUFO a JRE Ut a V rn OU JDU lt IJIOOr lt otite OVUXIXUIL LIIVO JEVDUUU JUUUXIE J DUUUUF rLruuu Ur uru DUFRUUERX UUFUUC 2 u ur IOVUUDVU lt JEUXTUUXF OULULLA UUUr uu LIVIE JOXUUUE UUXIUUX VUUUER lt TUUFUVE VERFOF EUXIEH lt JUH ORL JU Fr E Ivi lt lt v o o u INTEGRATED DNA TECHNOLOGIES qPCR Application Guide Experimental Overview Protocol Troubleshooting Fourth Edition qPCR Application Guide Experimental Overview Protocol Troubleshooting Fourth Edition Managing Editors and Contributors Nicola Brookman Amissah PhD Hans Packer PhD Ellen Prediger PhD Jaime Sabel Contributors Stephen Gunstream Jan Hellemans PhD Aurita Menezes PhD Brendan Owens Scott Rose PhD Rick Sander Jo Vandesompele PhD Integrated DNA Technologies Biogazelle NV Belgium Ghent University Belgium ID INTEGRATED DNA TECHNOLOGIES WWW IDTDNA COM q
28. Quantification cycle Cycles reaction 65 Incorrect 70 D Data analysis 36 46 52 qbasePls software 85 Design tools 82 See also Assays Design tools DNaseAlert Kit 66 dNTPs 44 Double quenched probes 25 Dye s Choice 69 90 HEX dye 14 25 33 35 lowa Black dye 25 26 TET dye 14 25 33 35 E Efficiency 27 34 36 46 49 55 56 67 68 70 Decrease over time 70 Effect of inhibitors 68 Effect on Eg 70 Efficiency correction 56 Emission 34 36 52 Enzymes 22 34 74 Avoiding contamination 19 Contamination 74 Multiplexing 34 Polymerase 42 44 49 73 Reverse transcriptase 21 64 Excitation 36 Exponential region 7 8 53 Extension times 23 30 64 F Fluorophores Absorbance ranges 35 FRET probes 11 See also Hybridization probes G gBlocks Gene Fragments 38 40 48 66 H HEX dye 14 25 33 35 Housekeeping genes See Reference genes Hybridization probes 11 See a so FRET probes l Inhibitors 68 74 Reaction 68 74 RNase 18 21 23 Instrument Calibration 71 73 Contamination 77 Settings incorrect 64 70 Intercalating dyes 13 Cyto dye 13 EvaGreen dye 13 91 AXCXIDI INTEGRATED DNA TECHNOLOGIES LC dye 13 Melt curves 46 SYBR Green dye 13 lowa Black dye 25 26 L Length Amplicon 30 49 Primer 28 29 31 Probe 28 29 Limit of detection LOD 50 Below 66 Limit of quantification LOQ 50 Linear range 50 Linear region 7 8 M Master mixes 35 43 45 65 72 80 Contamination 71 Correc
29. R Application Guide 1 2 4 Scorpions Probes Scorpions probes consist of a primer covalently linked to a spacer sequence followed by a probe that contains a fluorophore and a quencher Figure 5 A Figure 5 Scorpions Probes During the annealing step the primers hybridize in a sequence dependent Om manner to the complementary DNA strand The probe attached to one of the primers remains in a hairpin structure with the fluorescent dye D and quencher Q in close proximity the quencher absorbing the fluores rr AAAK A cence energy emitted by the dye In the extension step the polymerase begins DNA synthesis extending from the 3 ends of the primers As extension continues and the complementary sequence is synthesized the loop sequence of the probe hybridizes to the complemen O YA tary target sequence of the newly synthesized strand This separates the probe dye and quencher so that the quencher no longer absorbs the energy emitted by the dye The fluorescence is detected by the real time PCR instrument e he probe contains an amplicon specific complementary target sequence in the loop portion of the stem loop a spacer sequence a fluorophore dye D and an internal quencher O all contiguous with the primer e When not bound to the target the probe remains in a stem loop structure which keeps the quencher and fluorophore dye proximal and allows the quencher to ab sorb the fluorescence energy emitted
30. al master mixes optimized to work in most standard assays It is important to note that small changes in the master mix formulation can significantly affect assays and some formulations are designed for use with specific cycling conditions that is standard or fast cycling conditions A brief description of the role of the different components in a master mix is given below Buffer required to maintain optimum pH and salt conditions MgCl required to stabilize primer and probe interactions with DNA and as a co factor for Taq polymerase Occasionally it may be necessary to add more MgCl to the master mix to achieve optimum amplification results dNTPs the building blocks for DNA synthesis Some master mixes include dUTP and UNG enzyme to eliminate carryover of product from a previous PCR However dUTP is not incorporated as efficiently as dTTP and might affect amplification UNG Uracil N glycosylase enzyme used to eliminate PCR carryover contamina tion by degrading any PCR product with incorporated uracil bases If a master mix includes dUTP it is necessary to adapt cycling conditions to include an UNG step to degrade any previous PCR product Polymerase master mixes normally contain modified DNA polymerases to elimi nate nonspecific priming that may occur before the initial denaturation step Hot Start polymerases have been modified through use of an antibody or chemical modification or aptamers to be inactive at low
31. al amplification 56 Homologous sequences 46 Melt curves 46 Multiplex qPCR 34 No amplification 64 67 No amplification control 38 Off target 25 Outside linear range 50 Preamplification 28 Threshold 53 54 Amplification Plot Good curves examples 59 Poor curves examples 59 63 Relationship to Rn 52 Analysis tools 26 OligoAnalyzer Tool 26 29 80 84 UNAFold Tool 26 Annealing temperature 28 Incorrect 65 Annealing times 64 Assays Design 27 Limited samples 27 SNPs Single nucleotide polymorphisms avoiding 27 Design tools PrimerQuest Design Tool 14 83 PrimeTime Predesigned qPCR Assay Selection Tool 26 28 82 83 RealTime PCR Design Tool 26 83 Intercalating dye 14 46 5 Nuclease 27 38 Validation 46 B Baseline 7 8 52 53 64 68 69 72 81 Incorrectly set 68 69 Rising 81 BLAST analysis 34 67 85 C Calibration Dye 36 Instrument 36 73 CDNA 21 22 22 42 47 48 64 66 68 71 73 Constant volumes 42 Effects of sample preparation methods 68 Genomic contamination 71 See also Con tamination Mitigation Limited 27 66 67 Poor RNA quality 73 Reaction inhibitors 68 Reverse transcription 64 Storage 22 Contamination 66 71 77 See also Nucleases Mitigation 19 Controls 22 Negative 22 38 No reverse transcriptase No RT or RT 22 Positive 38 57 66 AACg 55 Cg Quantification cycle Definition 53 AACg 55 Relationship to baseline 52 Relative quantification 54 Too high 67 AACt See AACg Ct Threshold cycle See Cg
32. and UV lights can be useful but are not ab solutely necessary Short term UV light treatment is only effective against live organisms and not against purified nucleic acid Water carboys are not recommended for long term water storage because mi croorganisms can thrive in these containers Multiple freeze thaw cycles of oligonucleotides in a buffered solution are often mistakenly thought to lead to their degradation In order to prevent this prob lem users are often advised to make small aliquots of the primer probe mixes However IDT has shown that PrimeTime Assays containing primer probe mix es are stable in buffered solutions for over 30 freeze thaw cycles Figure 8 Ali quots are useful if the stock solutions will be accessed frequently with pipettes that are potentially contaminated with nucleic acids aPCR Application Guide 0 Freeze Thaws Amplification Plot Amplification Plot i j i p 1 000 E 2 HA n ll 0 10 15 20 25 Cycle Detector FAM1 Plot ARn vs Cycle v Threshold 0 09675021 15 Freeze Thaws 30 Freeze Thaws Amplification Plot Amplification Plot Amplification Plot Amplification Plot 1 000 ARn 1 000 E 2 P 0 10 15 A IN 00 cot LLL 2L o 5 10 15 20 25 20 25 Cycle Cycle 30 35 30 35 Detector ram Piot ARn vs Cycle Threshold 0 0967 5021 Detector FAM1 gt Plot arn vs Cycle Threshold 0 0967 5021 Panel
33. as degraded Check the instructions for the master mix to verify proper storage Also check for expira tion dates to be sure the mix is still current Replace any expired reagents Make a new preparation of the template if necessary Incorrect primer concentration Check that the tubes containing the primers were spun down before resuspension and the contents diluted to the correct concentration In crease the concentration of primers in 50 nM increments and monitor the shape of the amplification curve for any improvement 7 3 3 Instrument Cycling temperatures or time parameters incorrectly set It is possible that the cycling temperatures and time parameters are not set correctly Check that parameters match the recommended cycling conditions for the master mix used Do not interchange cy cling protocols between fast and standard master mixes Guidelines are provided in the protocol in Section 4 3 2 Also make sure the fluorescence is being collected at the ex tension step 70 AXCXIDI INTEGRATED DNA TECHNOLOGIES 7 4 Excessive or Unexpected Signal 7 4 1 Instrument Calibration Signal detected in wrong channels There may be bleed over of dye signal into another channel making it difficult to obtain an accurate reading of signal for any given dye This is usually due to improper instrument calibration Ensure that the real time PCR instru ment is compatible with and calibrated for each of the dyes used in a qPCR experiment To det
34. ates Most studies require larger numbers of samples On the other hand when performed by experienced users large screening studies that include many samples and fewer replicates may suffice Y Y Normal Mutant 48h 72h 24h 48h 72h e Multiple biological replications e 2 RT preps for each sample 1 no RT control Ub gt e 3 technical replicates for each sample No template control for each qPCR assay tested Lil e qPCR for gene of interest Multiple references tested for stable 97 expression across experimental conditions Figure 14 qPCR Assay Setup Outline of the replicates and controls needed for an experi ment comprising two different samples examined at several time points after treatment In clude 3 5 biological replicates for each time point studied For each biological replicate studied perform 2 reverse transcription reactions RT and 1 with no RT RT control For each cDNA sample gener ated set up 3 technical repli cates for qPCR analysis Include a No Template Control for each gene analyzed to identify any signal due to contamination aPCR Application Guide 4 2 6b Negative Controls IDT recommends the following three negative controls The no template control is an absolute requirement for all qPCR experiments 1 A no template control NTC omits DNA from the PCR This reaction serves as a gen eral control for unwanted nucleic acid contamination or pr
35. ation 4 2 2 Primers and Probes Use the PrimeTime Predesigned Assay Selection Tool to identify human mouse or rat assays We strongly recommend using the RealTime PCR Design Tool located on the IDT website for custom assays i e assays for genes of other species to be sure all of the important parameters are included in assay design IDT recommendations for primers probe and amplicon specifications are provided in the following text and summarized in Table 1 Primers Probe Amplicon Range Ideal Range Ideal Range Ideal Length 18 30 22 20 25 24 70 150 100 Melting o o Temperature 60 64 C 62 C 66 70 C 68 C NA NA GC content 35 65 50 35 6590 50 NA NA For probes that do not contain MGB Tm enhanced properties Table 1 Recommended Specifications for Primers Probes and Amplicons These specifications are based on a final reaction composition of 50 mM KCI 3 mM MgCl and 0 8 mM dNTPs 4 2 2a Primers Tm Typically an annealing temperature of 60 C is used during PCR The optimal melting temperature of the primers is between 60 and 64 C with an ideal temperature of 62 C which is based on the average conditions and factors associated with the PCR The melt ing temperature of the two primers should not differ by more than 4 C in order for both primers to bind simultaneously and efficiently amplify the product 28 AXCXIDI INTEGRATED DNA TECHNOLOGIES Length Aim for primer lengths of 18 30 bases with a balanc
36. ation to be measured while the reaction is occurring through use of a fluorescence detector in conjunction with the thermal cycler This allows analysis of the entire amplification curve rather than only the end point aPCR Application Guide Plateau Region an Figure 1 Phases of a Typical qPCR Amplifi cation Plot qPCR amplification plot phases include baseline exponential linear and plateau regions of the amplification curve Exponential Region a Baseline Region O 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 Cycle 1 2 1 5 Nuclease Assay The 5 nuclease chemistry utilizes two primers and a hydrolysis probe and capitalizes on the exonuclease activity of Taq DNA polymerase 3 The DNA probe is non extendable and labeled with a fluorescent reporter dye D and 1 or 2 quenchers Q which are maintained in close proximity to each other while the probe is intact Figure 2 The presence of one of the quenchers at the 3 end prevents extension of the probe by the polymerase While the probe is intact the energy emitted by the reporter dye is absorbed by the quencher s Because all three components 2 primers and 1 probe must hybridize to the target this method allows detection of the PCR product with greater specificity and high er accuracy In addition different probes can be labeled with different fluorescent dyes enabling multiple targets to be simultaneously detected in a single reaction
37. ch as the IDT ZEN double quenched probes 4 Optimize the individual reactions and ensure that they each have an efficiency 9096 5 Validate the multiplex reactions by running a combined reaction alongside an in dividual reaction to ensure similar performance Compare the standard curves and verify that the Cg values are similar at both the high and low ends A good multi plex should have similar curves and similar limits of detection Figure 13 6 Optimize the multiplex reactions Limit the primers for targets expressed at a high level to a 1 1 primer to probe ratio Increase the primer to probe ratio for targets expressed at lower levels IDT offers custom primer to probe ratio options for PrimeTime qPCR Assays Increasing the amount of enzyme and dNTPs added to 34 AXCXIDI INTEGRATED DNA TECHNOLOGIES the reaction may be necessary we recommend doubling the amount of these reagents There are a few commercially available master mixes that are specially formulated for multiplexing Dye Dye Absorption max nm Dye Emission max nm FAM 495 520 TET 509 539 HE 538 555 JOE 529 555 MAX 524 557 ia 550 564 TAMRA 559 583 ROX 588 608 TEXASRED 598 617 TEX615 596 bie TYE665 645 665 5 648 668 Table 3 Absorbance Range of Fluorophores Amplification Plot FAM Singleplex n FAM Multiplex Figure 13 Multiplex PrimeTime qPCR Assays Each reaction was performed using
38. cle Number Cycle Number Figure 10 ZEN Dual Quenched Probes Provide Superior Sensitivity 5 FAM probes with five different quench ers ZEN lowa Black FQ ZEN IBFO Black Hole Quencher BHO Eclipse lowa Black FO IBFO and TAMRA were synthesized to target the ACTB locus qPCR was carried out in triplicate using each probe a primer pair for ACTB and 0 5ng of cDNA All reactions were performed using Applied Biosystems TaqMan Gene Expression Master Mix under standard cycling conditions on the Applied Biosystems 7900HT Real Time PCR instrument helated Products at IDT Scilools Web Tools There are several free design and analysis tools available on the IDT website PrimeTime Predesigned qPCR Assay Selection Tool Use to select assays for human mouse and rat targets e RealTime PCR Design Tool Use to design primers probes and assays for gene targets in other species OligoAnalyzer Tool Use to analyze oligonucleotide melting temperature hairpins dimers and mismatches e UNAFold Tool Use to analyze oligonucleotide secondary structure For more information and to use these free SciTools Web Tools see Section 8 2 1 and the SciTools webinar under the Support tab on the IDT website www idtdna com 26 AXCXIDI INTEGRATED DNA TECHNOLOGIES 4 2 5 Nuclease Assay Design 4 2 1 General Design Considerations Know your gene A well designed assay begins with an understanding of the gene of interest incl
39. clease degradation and allow for rapid sensitive detection of RNases or DNases ReadyMade Primers and Randomers IDT offers a number of primers and rando mers including random hexamers and Oligo dT primers that are pre made puri fied and ready to ship upon order For more information and to order these products visit the IDT website at www idtdna com AXCXIDI INTEGRATED DNA TECHNOLOGIES Guidelines for Maintaining a Contamination Free Workplace At some point most qPCR users will experience some level of template contamina tion Simple steps can mitigate the risk of accidental contamination that results in non informative data and thus wasted time and money Design a unidirectional process flow PCR setup should be done in a template free room using reagents that never come into contact with potential contami nation sources This means keeping enzyme mixes water primers probes pi pettes tubes filter tips and plates in a room where template is never isolated or stored When mixes have been made and dispensed into the wells move the plates to a new location for template addition If using robotics do not use the same platform for setting up PCR assay plates and isolating nucleic acids Regular decontamination of commonly used equipment is recommended es pecially pipettes and work surfaces Regular cleaning of non porous surfaces with a 596 bleach solution is encouraged Bench top hoods with HEPA filters
40. d be assessed Throughout the process it is imperative that the sample remain free of RNases and DNases Here we provide some suggestions for isolation quantification assessing quality and preventing nuclease contamination 2 1 Isolate RNA isolation can be performed using organic extraction methods TRIzol reagent Invitrogen QIAzol reagent Qiagen RNA STAT 60 Tel Test Inc guanidium salt based methods 2 or a variety of solid phase RNA isolation kits that are available com mercially from companies including Qiagen Life Technologies Ambion and Promega The best method will depend on your sample type and the amount of RNA available for harvesting For example small RNAs and miRNAs can only be efficiently isolated using organic extraction methods 13 while solid phase kits are the appropriate choice for high throughput processing It is important that the RNA be extracted from all samples using the same method and that the resulting RNA be of high quality Differences in either sampling or isolation methods can lead to unwanted variation between samples Surfaces and supplies should be free of RNases Some samples may require DNase treat ment to remove genomic DNA contamination This step may not be necessary if the assay is designed to span exon junctions and thus only contain only exonic sequences If the isolated RNA is not going to be used immediately it should be frozen at 20 C for short term storage for at most a
41. d in the protocol in Section 4 3 2 IDT suggests using a linearized plasmid that contains the target sequence as template because it can easily be quantified and amplified The target sequence can be ordered as a gBlocks Gene Fragment or an Ultramer Oligonucleotide from IDT see the Products Box end of Section 5 2 3 for more information When the reaction has been optimized and the LOD is known try amplifying the cDNA DNA using 10 100 ng cDNA per reaction If the DNA still does not amplify try a new CDNA DNA template preparation to rule out transcription or sample prep inefficiencies Also using a gene specific RT primer in the first strand cDNA synthesis can also be used to increase the amount of target for detection Not enough template If too little template was added to the reaction the polymerase may not have been able to amplify the target to a level above the limit of detection 66 AXCXIDI INTEGRATED DNA TECHNOLOGIES LOD of the assay Increase the amount of template in the reaction Preamplification of RNA selecting for poly A RNA or using a gene specific RT primer in the first strand CDNA synthesis can also be used to increase the amount of target for detection See Section 4 2 1 for more information helated Products at IDT RNaseAlert and DNaseAlert Kits These reagents are fluorescence quenched oli gonucleotide probes that emit a fluorescent signal only after nuclease degradation and allow for rapid s
42. d or absent fluorescence signal is due to inefficient amplification by the primers First check that the primers or assays were diluted correctly Next rerun an assay that has worked previously to rule out problems with the master mix or thermocycler If the problem is assay specific run a sample of the reaction product on a high resolution agarose or non denaturing acrylamide gel to see if a PCR product of the correct size is present If so the primers are likely working See Section 4 3 2 for a protocol Probe degraded or degrading during reaction See Section 7 7 High or Variable Back ground Nuclease contamination causing degradation of nucleic acids Use a reagent such as the IDT RNaseAlert or DNaseAlert Kit to check for nuclease contaminants IDT recommends that the probe be resuspended in nuclease free TE buffer 10 mM Tris pH 7 5 8 0 0 1 mM EDTA and stored in aliquots at 20 C See Section 4 3 1 for a resuspension protocol and Section 2 4 for more information on avoiding nuclease contamination 7 1 4 Sample Expression Target below the limit of detection Amplification signal will not be detectable if the target gene is absent or expressed below the limit of detection LOD for the assay To determine the LOD for a particular assay amplify a positive control across a range of concentrations from 1 to 100 000 copies per well Optimization of the reaction may be required to achieve low LOD Guidelines for optimization are provide
43. d runs Hoe ACE Y RUE RODE HEROD Owe ORD DO 38 4 3 PrimeTime qPCR Assay Protocol naaa a 40 4 3 1 Resuspension Protocol ooo 40 4 3 1a Avoiding Probe Degradation e 41 4 37 Assay ProtOCOl ue a xod fedem Daw does KV Rep SCRCN Oe M opor ee eas 42 A So OAS UCT MIKES eo anc NA a og CE wk oe e GOR wee SO we oe a ep oe RE S 44 S A ssdy VAIIGQUION su dw x aseo ARA ES YOR s 46 5 1 Specificity Analysis aoaaa ees 46 DARE 080 sarna Pe aoe AO Oe A be 8 46 5 1 2 Amplicon Size Analysis kak ee edo Ro Soo ee OK X ed a e 46 Dale CAUNO es iaa A TTC 46 5 2 Efficiency Analysis 47 mE amar COVES ama ra Be ee ee ee sd Be Bx 47 5 22 Range O DION suce vows ooo r3 5 Fob RODE DET SET ae BESS 245 47 UE MMC 48 5 3 PCR EfTICIGFICV iu aeos 245404400 445 6 903 XO UR ES GED OX RD qe 49 5 4 Limit of Detection LOD and Limit of Quantification LOQ 50 2 Biniesr Dynanme dale i re sese ok wx de 2 eee amp oe oe 039 A Son 50 56 Precisi n ana Vania DU yae ue ae a aa 38 rie we A RA ate Gh e AOR ae RR BR 51 O Data Analysis aw onc d d A deed ciar ee MPU ee ee ee 52 ORTAR manO RPU S a aaa a derek aa a ea hs hw Hea a A 52 6 2 Setting the Baseline onnaa aaa es 52 6 2 setting ihe TAresNOld S es a a cai qe e Te RO we diae ec Dente de Se se a a ee Be c 53 6 4 Determining Gene Expression Changes ee 54 6 4 1 Absolute Quantification ee 54 6 4 2 Relative Quantification ee 54 6 4 2a Normalization
44. de the IDT ZEN molecule as a secondary internal quencher allow longer probes to be used while pro viding strong quenching and increased signal See Section 4 1 3 for more information GC content Aim for a GC content of 35 6596 and avoid a G at the 5 end to prevent quenching of the 5 fluorophore As with the primers avoid sequences that may create secondary structures or dimers Location Ideally the probe should be in close proximity to the forward or reverse prim er but not overlap although this is not absolutely necessary Probes can be designed to bind to either strand of the target 29 aPCR Application Guide 4 2 2c Amplicons Length Design amplicons of 70 150 bp which will allow the primers and probe to compete for hybridization and provide a sequence that is long enough for all compo nents to bind This length is most easily amplified using standard cycling conditions Longer amplicons of up to 500 bases can be generated but cycling conditions will need to be altered to account for the increased extension time Amplicons or assay designs should span an exon exon junction to reduce the possibility of genomic contamination Tm Calculate all melting temperatures under real time PCR conditions standard pa rameters for qPCR are 50 mM K 3 mM Mg and 0 8 mM dNTPs however they can vary widely from this particularly with respect to Mg concentration See Section 4 2 2d be low for more information on calculating melti
45. e between the melting temperature purity specificity and secondary structure considerations GC content Ensure that the primers are specific to the target and that they do not contain regions of four or more consecutive Gs 17 The GC content should be within the range of 35 6596 with an ideal content of 5096 which allows complexity while still maintaining a unique sequence Avoid sequences that may create secondary structures self dimers and heterodimers use programs such as the IDT OligoAnalyzer tool to find potential sites that are likely to form these structures 4 2 2b Probes Tm The melting temperature of the probe should be 6 8 C higher than the primers and should fall within the range of 66 70 C for a standard two step protocol If the melting temperature is too low the probe will not bind to the target In this case the primers may amplify a product but as the probe is not bound to a target it will not be propor tionally degraded and thus will be unable to provide the fluorescence that is necessary to detect the product Length The length of a single quenched probe should be 20 30 bases to achieve an ideal Tm without increasing the distance between the dye and quencher such that the quencher will no longer absorb the fluorescence of the dye probes with a single ter minal quencher that are longer than 30 bases may perform poorly due to the distance between the quencher and dye Double quenched probes that inclu
46. e custom design tool also allows you to target specific exon locations and will allow you to view primer and probe sequences prior to purchase Alternatively if you are working with human mouse or rat genes IDT has predesigned assays covering those tran scriptomes that can be located using a simple searchable format see Section 4 1 2 below 4 1 2 PrimeTime Predesigned gPCR Assays IDT offers PrimeTime Predesigned qPCR Assays for human rat and mouse targets The IDT design engine incorporates numerous parameters optimized to yield a robust qPCR assay The design process uses up to date sequence information from the NCBI RefSeq database and accurate Tm and secondary structure prediction to protect against off target amplification and avoid SNPs SNPs that occur in 1 or more of the human popu lation are present in the genome on average every few hundred bases 16 Keeping up to date with the continually increasing number of annotated SNPs is critical for qPCR experimental success The frequently updated design engine used to generate Prime Time Predesigned qPCR Assays allows researchers to focus on the experiment rather than the design 4 1 3 ZEN Double Quenched Probes IDT has developed an internal ZEN quencher for the pro duction of double quenched probes that produce qPCR data with less background and increased signal Double quenched probes contain a 5 fluorophore choice of FAM HEX JOE TET or MAX a 3 IBFO quencher and an inte
47. e pairs 20 base pairs 30 base pairs 30 40 so Ty 60 70 80 90 Temperature C Predictive algorithms have recently been significantly improved the nearest neighbor method predicts Tm with a higher degree of accuracy than previously used methods 18 Older formulas which do not take into account interactions between neighboring base pairs provide Tm predictions that are too inaccurate for real time PCR design IDT scientists have published experimental studies on the effects of Na K and Mg on the stability of oligonucleotide duplexes and have proposed a model with greater accuracy 19 The linear Tm correction has previously been used to account for salt stabilizing ef fects but melting data from a large oligonucleotide set demonstrated that non linear effects are substantial and must be considered 19 Figure 12 Salt Concentration Affects Tm O The stability of a 25 bp duplex CTG GICTGG ind ATC TGA GAA CTT CAG G varies with K and Mg concentrations Competitive binding ET of ions to DNA is observed IM 100mM 10mM ky ImM 3 aPCR Application Guide PCR buffers also contain deoxynucleoside triphosphates dNTPs which bind magne sium ions Mg with much higher affinity than DNA Since they decrease the activity of free Mg Tm may be also decreased 19 Figure 12 The best predictive algorithm considers this effect as well IDT SciTools design tools see below employ the latest nearest nei
48. e probe containing the activated 3 donor fluorophore D is positioned to transfer its energy to a second probe containing a 5 acceptor fluorophore A The fluorescence of the acceptor is re o P corded by the instrument In the extension step the polymerase synthesizes new DNA strands by extend 3 ing from the 3 ends of the primers o 2 The 3 end of the acceptor probe contains a phosphorylation modification that pre vents extension of the probe during amplification Since the 3 end of the donor probe is labeled with a fluorophore no phosphorylation is required to block exten sion on this probe During the annealing step the primers and both probes hybridize to the target with the probes in a head to tail configuration which brings the two fluorophores close together A light source is used to excite the donor fluorophore which then excites the accep tor reporter fluorophore through FRET Fluorescence Resonance Energy Transfer A detector is set to read the emission wavelength of the acceptor fluorophore In order for the energy transfer to occur the spectra of the two fluorophores must overlap so that the donor fluorophore can excite the acceptor fluorophore These types of probes require dedicated machinery in order to excite the donor fluorophore The LightCycler Real Time PCR System Roche is designed for such probes IDT synthesizes the probes and primers needed for this system aPC
49. ensitive detection of RNases or DNases For more information and to order these products visit the IDT website at www idtdna com 7 2 Low or Delayed Signal 7 2 1 Design Specificity The Cy is higher than the expected value This observation likely indicates low primer efficiency There may be mismatches between the target and primer probe sequences Determine the amplification efficiency by using a serial dilution of a plasmid contain ing the target sequence Perform a BLAST search to confirm the specificity of the target and assay sequences See Section 8 3 1 for more information about this free NCBI tool Run a gel to see if the correct size of product is being amplified If you are not using a predesigned assay from IDT check that your primer and probe do not span a SNP site Note that all PrimeTime qPCR Assays are tested with up to date sequence information specifically to avoid assay design over SNP sites thus preventing this problem 7 2 2 Reaction Setup Primers or probe were not completely resuspended See Figure 22 for an example of curves from a reaction run with a low concentration of probe Suboptimal primer or probe concentration is most often due to incomplete resuspension Check the resus pension protocol in Section 4 3 1 and confirm that these reagents were properly resus pended Calculation or dilution errors can also result in too little probe primer A less likely possibility is that the recommended primer and probe concentrat
50. entration 77 79 Insufficient 72 TAMRA diminished signal 78 RT qPCR 22 23 27 See also qPCR Reverse transcription protocol 23 S SciTools web tools 14 82 Scorpions Probes 12 Quencher 12 93 AXCXIDI INTEGRATED DNA TECHNOLOGIES Slope Effect of inhibitors 74 PCR efficiency 47 49 SNPs Single nucleotide polymorphisms 27 Specificity 46 Amplicon size analysis 46 BLAST analysis 67 70 Melt curves 46 Sequencing 46 Standard Curves Absolute 47 gBlocks Gene Fragments use of 48 Range of Dilution 47 Relative 47 Template 48 Ultramer Oligonucleotides use of 48 Storage CDNA 22 Oligonucleotides 41 Probe 41 T TAMRA Diminished signal 78 Emission wavelength 78 Taq polymerase 44 80 Template Excessive 72 Insufficient 66 TET dye 14 25 33 35 Threshold 53 Tm See Melting temperature Tm Two step RT qPCR 22 23 27 29 U Ultramer Oligonucleotides 38 40 48 66 Uracil N glycosylase UNG 44 V Validation 46 Variability 51 Z ZEN Double Quenched Probes 25 Overcome poor quenching 76
51. ermine if calibration of your instrument is required run assays with each dye in dividually and use the software to determine whether dyes other than that expected show up in a particular channel e g run an assay with a Cy5 labeled probe and look at the other dye channels to see whether there is bleed over signal Erroneous signal in a channel is indicative of poor calibration To calibrate your instrument refer to the manu facturer s protocol for your instrument All instruments should have monthly maintenance including calibration Refer to the instrument manual for instructions Run an assay that has previously worked to see if it is still working 7 4 2 Assay Specificity Non specific assay Unexpected or excessive expression may mean the assay is not spe cific for the target transcript or is detecting additional transcript variants e g alterna tively spliced forms Using a second PrimeTime Predesigned qPCR Assay located in a different region of the target will verify the results or uncover an artifact 7 4 3 Contamination Reagent Contamination It is possible that the master mix or other reagents have been inadvertently contaminated with the amplicon See page 19 for additional ways to pre vent contamination Genomic Contamination Amplification occurring in the no RT control may indicate ge nomic contamination This may also result in higher than expected expression in your samples If genomic DNA is not your template of i
52. ers and molecular beacon probe hy A bridize in a sequence dependent manner to the tar get DNA Hybridization of molecular beacons to the complementary target separates the fluorescent dye D and quencher Q so that the quencher no longer absorbs the energy emitted by the fluorophore The resulting fluorescence is detected by the real time PCR instrument Molecular beacons in excess of the target DNA sequence reform the hairpin during the anneal ing phase and the quencher absorbs the fluorescence emitting the energy as heat In the extension step the polymerase begins DNA synthesis extending from the 3 ends of the primers When the polymerase reaches the molecular beacon the molecular beacon is dis placed without being degraded Thus probe can par ticipate in multiple rounds of annealing Meanwhile the polymerase continues extension of the primers to complete synthesis of the DNA strand ds AXCXIDI INTEGRATED DNA TECHNOLOGIES 1 2 3 Hybridization FRET Probes Hybridization FRET probes consist of two separate fluorescent dye labeled probes one probe is labeled on the 3 end with a donor fluorophore D and the other is labeled on the 5 end with an acceptor fluorophore A Figure 4 0 OR A Figure 4 Hybridization FRET Probes During the an p nc nealing step the primers and probes hybridize in a sequence dependent manner to the complementary 43 DNA sequence Th
53. exing 35 Predesigned assays 25 Protocol 42 Resuspension 40 Stability to freeze thaw 20 Probes Degradation 66 76 77 GC content 29 Integrity 76 Length 29 Location 29 Melting temperature Tm 29 Too much 72 Q qbasePtus software 85 qPCR 7 Amplicons 30 Baseline setting 52 Limit of detection LOD 50 Limit of quantification LOQ 50 Multiplex 34 Negative controls 38 One step 22 Positive controls 38 Precision 51 Primers 28 Probes 29 Replicates 36 RFU 52 Rn 52 Threshold setting 53 Two step 22 Variability 51 Workflow 15 Qualitative analysis 58 Quality 17 Quantification Absolute 54 Relative 54 Quencher 8 10 12 26 76 Distance from probe 29 Poor quenching 76 ZEN quencher 25 26 92 R Reaction efficiency 49 Reaction parameters 73 See also Cycles reaction Reaction setup No master mix 72 Pipetting errors 72 Poor mixing 72 Real time PCR 7 See also qPCR RealTime PCR Design Tool 83 Reference dye 44 See also ROX Concentration 77 Reference genes 55 Relative quantification 54 Efficiency correction 56 Normalization 55 Replicates 36 Inconsistent 72 Reporter dyes Choice of 32 Instrument compatibility 33 Reverse transcription 21 Choice of primers 21 Nonoptimal 64 Protocol example 23 Sample quantity 21 RFU Relative fluorescence units 52 RNA Integrity 17 Isolation 16 Quality 16 73 Quantification 16 RNase 18 66 67 RNaseAlert Kit 66 Rn Normalized reporter signal 52 ROX 44 Incorrect conc
54. experiments as well as edu cational materials and courses For a complete overview of Biogazelle wet lab and data mining services see www biogazelle com 85 aPCR Application Guide 9 References 1 Bustin SA Benes V et al 2009 The MIQE Guidelines minimum information for publication of quantitative real time PCR experiments Clin Chem 55 4 611 622 Updates Bustin SA Beaulieu JF et al 2010 MIQE pr cis Practical implementation of minimum standard guide lines for fluorescence based quantitative real time PCR experiments BMC Mol Biol 11 74 78 and Bustin SA Benes V et al 2011 Primer sequence disclosure A clarification of the MIQE Guidelines Clin Chem 57 919 921 Chirgwin JM Przybyla AE et al 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease Biochem 18 5294 5299 Heid CA Stevens et al 1996 Real time quantitative PCR Genome Res 6 10 986 994 Hindiyeh M Levy V et al 2005 Evaluation of a multiplex real time reverse transcriptase PCR assay for detection and differentiation of influenza viruses A and B during the 2001 2002 influenza season in Israel J Clin Microbiol 43 2 589 595 Thiel CT Kraus C et al 2003 A new quantitative PCR multiplex assay for rapid analysis of chromosome 17p11 2 12 duplications and deletions leading to HMSN HNPP Eur J Hum Genet 11 2 170 178 Nazarenko Pires R et al 2002 Effect of primary and secondary struc
55. f local simi larity between sequences It calculates the statistical significance of matches and can be used to select primers and probe sequences for qPCR assays However due to the heuristic nature of BLAST and removal of low complexity data queries for such short sequences often return incomplete data See the DECODED 1 1 April 2011 newsletter article Tips for Using BLAST to Locate PCR Primers for recommendations on how to use this tool 8 3 2 RT qPCR Data Analysis The use of dedicated software such as the qbase 5 software from Biogazelle avail able at www qbaseplus com for the analysis of your RT qPCR data can speed up data analysis minimize errors created by manually entering data and formulas and simplify reporting of data analysis methods in accordance with MIQE guidelines 1 The qbasePtUS software is an RT qPCR analysis package that meets the MIOE guidelines The software allows direct import of Ca values data tables from qPCR instruments from a variety of manufacturers and provides algorithms for removal of data errors normal ization of data to one or more reference genes and correction of inter run variation using inter run calibrators The qbase 5 software also offers statistical tools for RT qPCR data analysis and tools for graphical presentation of analyzed data In addition to the qbase 5 software Biogazelle offers a variety of other services to help investigators with design and implementation of RT qPCR
56. f minimum standard guidelines for fluorescence based quantitative real time PCR experiments and Primer Se quence Disclosure A Clarification of the MIOE Guidelines provide additional guidance on how to implement the MIOE standards in practice with additional information on sample handing experimental conditions normalization to reference genes and data analysis 1 8 2 IDT Resources in Print and Online 8 2 1 SciTools PCR Assay Design Tools IDT offers a variety of free design and analysis tools in the online SciTools suite For RT qPCR designs using 5 nuclease assays the suite contains tools for selecting PrimeTime Predesigned qPCR Assays and designing PrimeTime Custom qPCR Assays In addition the SciTools suite of analysis tools contains convenient dilution calculators and tools for analyzing oligonucleotide properties The SciTools software described in this section are all freely available at www idtdna com under the SciTools tab There is also an excellent webinar see Section 8 2 3 under the Support tab that demonstrates how to use these SciTools programs 82 AXCXIDI INTEGRATED DNA TECHNOLOGIES 8 2 1a Predesigned gPCR Assays Tool The IDT Predesigned qPCR Assays tool is a dedicated design tool for the PrimeTime Pre designed qPCR Assays If your target is a human mouse or rat sequence this program offers the highest level of bioinformatics analysis including avoidance of known SNPs BLAST search to avoid cross react
57. f the ROX signal is too high the ROX signal will be higher than FAM in the multicom ponent plot and the ARn will be low Figure 23 If samples are not sealed properly the effective ROX concentration measured by the instrument increases due to evaporation which increases the number by which your samples will be normalized and results in incorrect Cg values 7 8 2 Higher than expected or noisy amplification curves low ROX ROX degradation or low ROX concentration can cause the ARn to be high and noisy as the instrument tries to normalize data using a weak inconsistent ROX signal See Figure 23 T aPCR Application Guide 7 8 3 Amplification curve drops off and has an atypical shape Evaporation Passive reference signal fluctuation can be caused by evaporation if sam ple tubes or plates are not sealed properly Ensure secure sample tube plate closure Inconsistent dye concentration Fluctuations of ROX signal within an experiment can result in invalid gene expression data Ensure ROX does not vary significantly within an experiment by looking at the raw background signal before the fluorescence data has been normalized 7 8 4 When used with ROX the TAMRA signal is diminished TAMRA has an emission wavelength close to the absorption wavelength of ROX There fore when probes labeled with TAMRA are used in reactions with master mixes that contain ROX the signal from TAMRA can be greatly diminished This can affect the nor malized
58. fect of differ ing PCR efficiencies becomes more apparent with increasing cycle number 55 aPCR Application Guide ACg Cg target Ca reference equation 1 AACg ACg sample ACg control equation 2 R 2 44Cq equation 3 1 Determine the Cg value for each reaction 2 Calculate the difference in Cg values for the gene of interest target and the endog enous control s reference This is the ACg as shown in equation 1 3 Subtract the control condition ACg from the treated condition ACg to find the AACq as shown in equation 2 4 To calculate the ratio of gene expression change R of the sample relative to the control the negative value of this subtraction AACg becomes the exponent of 2 and represents the difference in the corrected number of cycles to threshold The value of 2 is used because the assumption is that the product doubles in each cycle This is shown in equation 3 5 For example if the control sample ACg is 2 and the treated sample ACg is 4 then 2 AACg will be 0 25 Therefore the level of the gene of interest in the experimental sample is 2596 of that in the control sample This can be verified by looking at the qPCR curves the experimental sample would require additional cycles to produce the same amount of product as the control sample because of the lower starting amount of the transcript being measured 8 28 31 6 4 2b Efficiency Corrected Gene Expression Measurements To accoun
59. g BRCA1 GAPDH BRCA1 GAPDH Replicate 1 252 14 1 Control Cg Replicate 2 257 13 8 254 14 0 Replicate 3 25 4 14 2 Replicate 1 22 1 13 5 Treated Cg Replicate 2 225 13 9 223 13 7 Replicate 3 22 3 o Table 7 Ca Values for Determining Relative Quantification oF aPCR Application Guide Using equation 4 the researcher was able to calculate the gene expression fold change between experimental and control samples ACg_BRCA1 Eg control Cg treated ACg_BRCA1 254 223 ACg_BRCA1 3 1 ACg_GAPDH Cg control Ca treated ACg_GAPDH 14 0 13 7 ACg_GAPDH 0 3 1 0 93 1 0 98 03 R 6 25 Therefore in the experimental sample BRCA1 expression levels increased 6 25 fold rela tive to the control sample A more accurate measurement was made by taking the am plification efficiency of each assay into account If equation 3 which assumes 100 assay efficiency had been used instead the result would have been 6 96 which would have been an overestimation of the fold change in gene expression This example is a simplistic analysis that uses a single reference gene In practice at least 2 stably expressed reference genes are recommended For a more detailed outline see Hellemans et al 26 6 5 Qualitative Analysis For some applications the purpose of PCR may be to determine the presence or absence of a particular nucleic acid target rather than to compare MRNA levels among samples For these applicat
60. g expression levels were northern blotting RNase protection assays or traditional endpoint reverse transcription RT PCR Endpoint RT PCR was an improvement over the older methods due to its ease of use and the much smaller amounts of RNA needed for the reaction However with this method expression levels can only be observed by performing agarose gel electrophoresis on a sample of the product at the end of the entire reaction While traditional RT PCR can be useful for determining the presence or absence of a particular gene product qPCR has the advantage of measuring the start ing copy number and detecting small differences in expression levels between samples qPCR allows investigators to observe PCR product accumulating over the entire am plification curve and eliminates the need to run a gel which reduces the duration of the process and the chance of contamination Amplification and quantification occur simultaneously A typical qPCR amplification plot has baseline exponential linear and plateau phases Figure 1 Amplification reaches a plateau as the reaction components are exhausted and PCR products self anneal and thus prevent further amplification In endpoint PCR amplification can only be viewed at the end of the reaction and only the final plateau is observed any differences in initial abundance are obscured In contrast qPCR quantifies the PCR products while the amplification is in progress Fluorescent re agents allow amplific
61. g real time RT PCR Nat Protoc 1 3 1559 1582 86 22 23 24 25 26 2 28 29 30 87 AXCXIDI INTEGRATED DNA TECHNOLOGIES The International HapMap Consortium 2007 A second generation human haplotype map of over 3 1 million SNPs Nature 449 851 861 Poon K and Macgregor RB Jr 1998 Unusual behavior exhibited by multistranded guanine rich DNA complexes Biopolymers 45 6 427 434 Owczarzy R Vallone PM et al 1997 Predicting sequence dependent melting stability of short duplex DNA oligomers Biopolymers 44 3 217 239 Owczarzy R Moreira BG et al 2008 Predicting stability of DNA duplexes in solutions contain ing magnesium and monovalent cations Biochem 47 19 5336 5353 SantaLucia J Jr and Hicks D 2004 The thermodynamics of DNA structural motifs Annu Rev Biophys Biomol Struct 33 415 440 McTigue PM Peterson RJ and Kahn JD 2004 Sequence dependent thermodynamic param eters for locked nucleic acid LNA DNA duplex formation Biochemistry 43 18 5388 5405 Xia T SantaLucia Jr J et al 1998 Thermodynamic parameters for an expanded nearest neighbor model for formation of RNA duplexes with Watson Crick base pairs Biochemistry 37 42 14719 14735 Nolan T Hands RE et al 2006 SPUD a quantitative PCR assay for the detection of inhibitors in nucleic acid preparations Anal Biochem 351 2 308 310 Eisenberg E and Levanon EY 2003 Human Housekeeping ge
62. ge Cq Val m 0 08 ng cDNA YNNN NSN N N Ni OE DO D UO N o D O h 000000000000 Amplification Plot 10 Minutes Amplification Plot 504 354 1 000 Cycle 1 1 1 1 1 20 25 30 35 40 45 50 55 60 65 Detector ff ami Plet aRn vs Cycle 7 Thresholet 0 30115252 ime sei Figure 21 Poor Sample Quality Delays Cg To demonstrate the effect of poor RNA quality on Cg values 8 ng 0 8 ng and 0 08 ng RNA were left untreated intact or were treated with NaOH for 2 min 5 min or 10 min to create increasing levels of degradation The electropherograms show samples of A intact RNA and B degraded RNA Cq values were delayed for the degraded RNA compared to the intact RNA sample as shown by C the average Cq values and D the amplification curves 4 AXCXIDI INTEGRATED DNA TECHNOLOGIES 7 7 High or Variable Background 7 7 1 Reaction Setup Addition of too much probe High probe concentration is usually due to errors in dilu tion or reaction setup Refer to the protocol to verify that probe was added and at the appropriate concentration See Section 4 3 2 for a protocol Also ensure that calculations for the dilution of probe are correct and that the probe was diluted appropriately Less likely is that the recommended primer and probe concentrations are not ideal for your sample This can be tested by decreasing the concentration of probe stepwise by 25 nM Amplification Plot A
63. ghbor method thermodynamic parameters 20 22 and the improved salt effects model 19 to achieve state of the art predictions of melting temperatures with an average error of approximately 1 5 C helated Products at IDT Scilools Design Tools A number of free design and analysis tools are available on the IDT website These include the RealTime PCR Design Tool for designing prim ers probes and assays OligoAnalyzer Tool for analyzing oligonucleotide melting temperature hairpins dimers and mismatches and mFold for analyzing the sec ondary structures of oligonucleotides For more information and to use these free SciTools programs visit www idtdna com 4 2 3 Choosing the Correct Reporter Dye for the Instrument The choice of fluorescent dye will depend on the instrument you are using and the com patibility of the dye with the instrument Table 2 lists dyes recommended by IDT that are compatible with common real time PCR instruments Refer to your instrument manufac turer s guidelines for information specific to your particular instrument FAM is the most popular of these dyes and is often more sensitive than some of the other dyes available 32 XIDT INTEGRATED DNA TECHNOLOGIES Instrument Compatibility with Reporter Dyes ae Siz pileg Applied Biosystems 7000 StepOne Plus e OOl X oe Rx x x x Applied Biosystems 7500 Fast e X oO Rio e O Applied Biosystems 7900
64. he enzymes RNase inhibitor to prevent RNA degradation and a reverse transcriptase enzyme 3 1 Sample For accurate comparison in the qPCR step equal amounts of starting RNA should be used from each sample in the RT reaction Large variations in the amount of RNA be tween RT reactions can lead to fluctuations in RT efficiency Poor RT may lead to loss of signal or failure to detect transcripts with low levels of expression 3 2 Choice of Primers The type of primers used will depend on the experimental goal Both random primers and oligo dT primers will produce random cDNA while gene specific primers will pro duce cDNA for a specific target Random hexamer and nonamer primers bind to RNA at a variety of complementa ry sites and lead to short partial length cDNAs These primers can be used when the template has extensive secondary structure Random primers will produce the greatest yield but the majority of the cDNA will be copies of ribosomal RNA unless it is depleted prior to RT PCR The main advantage to using random primers is the preservation of the transcriptome so that any remaining cDNA can be used in other qPCR assays The disad vantage is that low abundance messages may be under represented due to consump tion of reagents during cDNA synthesis of the more prevalent RNAs Random hexamers produce a greater amount of cDNA while random nonamers produce longer products Gene specific oligonucleotide primers which selectively pri
65. his data can be made with confidence 5 5 Linear Dynamic Range The linear dynamic range is the range over which a reaction is linear as established by the standard curve This range is defined by the higher and lower limits of quantification see LOQ in Section 5 4 Amplification measured outside this range cannot be quanti fied accurately with high levels of confidence Ideally the curve should span 5 6 logo concentrations and at a minimum span 3 4 orders of magnitude with 4 5 data points It is important to ensure gene expression calculations are made within the linear dy namic range 50 AXCXIDI INTEGRATED DNA TECHNOLOGIES 5 6 Precision and Variability The precision of an experiment depends on many factors Results can vary with tem perature changes which cause annealing or denaturation differences in concentration pipetting errors and stochastic variation 1 Precision can be determined by performing sample replicates within an experiment and between experiments 1 Sometimes normal biological variation between samples may exceed the variation in gene expression Such variation must be taken into consideration when designing the experiment and analyzing the data See Willems et al 27 for examples of how to work with variable samples 51 aPCR Application Guide 6 Data Analysis This section provides general guidelines for qPCR data analysis For assistance with data analysis we recommend using a software
66. ible program can be of great use IDT Technical Support is available to offer assistance with this program to help you meet your specific design challenges 63 aPCR Application Guide 8 2 2 Additional SciTools Software In addition to assay design tools the list of available SciTools programs includes cal culators and oligonucleotide analysis tools that may be helpful for setting up RT qPCR experiments especially when working with custom assays The OligoAnalyzer tool is the most heavily used of the IDT SciTools programs The pur pose of the OligoAnalyzer tool is to analyze the properties of input oligonucleotide se quences It provides information about secondary structures such as hairpin and primer dimer formation as well as Tm GC content effects of modifications or buffer conditions on those properties and an assortment of other useful information that can affect RT qPCR or other application performance The DilutionCalc tool is an easy to use calculator designed to compute the volume of concentrated oligonucleotide stock required in order to achieve a desired dilution vol ume and concentration Similarly the ResuspensionCalc tool calculates the volume of buffer or water to add to a dry or lyophilized oligonucleotide to reach a desired final concentration 8 2 3 Webinars IDT produces informational webinars to guide researchers through RT gPCR experi mental design and setup Previous webinars are archived at www idtdna co
67. ies present in the sample cycling conditions and the enzyme used 25 The precision of a particular PCR assay is depen dent upon the efficiency of the reaction Current quantification models do not allow for efficiency correction between samples However assay specific efficiency variation can be corrected for when calculating differences in gene expression Such correction is required for obtaining the most accurate results See Section 6 4 2 for more information on efficiency corrections The efficiency of a successful assay will be between 90 and 110 Amplification effi ciency can be calculated by analyzing the slope of the log linear portion of the standard curve When the logarithm of the initial template concentration is plotted on the X axis and Ca is plotted on the Y axis PCR efficiency 107 sore 1 1 Based on this equation 49 aPCR Application Guide the theoretical maximum of 1 00 100 indicates a doubling of the product with each cycle Efficiencies derived from dilution series are not exact values but estimates with an uncertainty This explains apparently impossible efficiencies of 10096 and suggests that an assay with an efficiency of 9496 is not really different from one with an efficiency of for example 9996 Hellemans et al 26 presented formulas to determine the error uncertainty in the estimated PCR efficiency derived from a standard curve Biogazelle qbaseP uS software automatically calculates these erro
68. ike this Intact BN Threshold Partially degraded XIDT INTEGRATED DNA TECHNOLOGIES Your initial dilutions higher concentrations for the stan dard curve are not producing expected Ca intervals See Section 7 6 4 You are observing later than expected Cg values and or inconsistent replicates You may be experiencing Nucleic acid degradation See Section 7 6 4 Pipetting errors See Section 7 6 1 aPCR Application Guide 7 1 Little to No Amplification 7 1 1 Reaction Setup Omitted or incorrectly diluted reaction component It is possible that one of the compo nents was inadvertently left out or added at an incorrect concentration We recommend that you repeat a failed experiment once to make sure it was not due to a simple mistake at the first attempt Refer to the protocol to verify that all listed components were added and at the appropriate concentration See Section 4 3 2 for a protocol Expired reagent Check the expiration date on all reaction components to make sure they are not expired Replace any expired reagents Incorrect instrument settings The baseline fluorescence should be above background The absence of fluorescence signal may be due to a problem with the instrument such as an incorrect filter setting Run a reaction without the primer probe mixture and com pare it to an experimental plate to see if the experimental plate is producing any fluores cence above background 1 2 Reaction Parameter
69. imer dimer formation that may make the results more difficult to interpret particularly when using SYBR Green dye Perform this control for each assay A no reverse transcriptase control CRT omits the reverse transcriptase in the re verse transcription step of a qRT PCR The purpose of this control is to assess the amount of genomic DNA contamination present in an RNA preparation Perform this control for any assay that might amplify genomic DNA A no amplification control omits the DNA polymerase from the PCR This reaction serves as a control for background fluorescence of the PCR assay and probe stability 4 2 6c Positive Controls An exogenous positive control is external DNA or RNA carrying a target of interest These control reactions will alert you to any components in the sample that might inhibit reverse transcription and or PCR IDT synthesizes gBlocks Gene Fragments MiniGene Synthetic Genes and Ultramer Oligonucleotides which can serve as exogenous positive controls with known starting copy number 23 An endogenous positive control is a native target that is present in the experimen tal sample of interest and can serve as a normalizer among samples These control reactions will correct for quantity and quality differences between samples IDT rec ommends that you test at least two but preferably three normalizing or reference genes to ensure accurate internal controls The most appropriate normalizing gene to u
70. ion and off target amplification see Section 8 3 1 and recognition of splice variants The program is regularly updated with current sequence information from NCBI ensuring assays are designed with up to date bioinformatics 8 2 1b RealTime PCR Assay Tool The IDT RealTime PCR tool provides effective qPCR assay design through a user friendly interface for targets other than human mouse and rat and is the recommended tool for designing custom assays The program is customizable at many levels and can be used to design oligonucleotides for qPCR assays with or without probes This is useful if you plan to run qPCR assays using SYBR The tool also includes the ability to direct the assay to specific regions of target such as select exon junctions or a particular exon found in a splice variant GPCR conditions can be specified within the program as can primer probe and amplicon parameters If no customization is needed this program can quickly access current NCBI data and recommend an assay for your qPCR target 8 2 1c PrimerQuest Design Tool The PrimerQuest design tool is not a dedicated qPCR assay design program However it is highly customizable and useful for the design of qPCR assays with non standard requirements For example you can use this design tool to direct the assay towards specific regions of your target or you can specify primer or probe sequences So if your design requires more demanding customization this highly flex
71. ions Section 4 3 2 are not optimal for your sample This can be tested by adjusting the concentration of primers and probe in 25 nM increments 67 aPCR Application Guide 7 2 3 Sample Expression Inhibitors in reaction Running several dilutions of the cDNA template can be used to determine the presence of inhibitors that may be limiting target amplification If increas ing template cDNA concentration does not lead to a linear increase in signal it is likely that inhibitors are present in the sample Also if there are inhibitors present in the sam ple the highest template concentration will contain the highest concentration of inhibi tors Amplification efficiency will be reduced and 10X dilutions will not be separated by 3 32 cycles Make a new preparation or repurify the cDNA template if this is the case Sample prep method skews target representation Due to inconsistencies in sample iso lation methods cDNA preparations can have differing amounts of transcript ends Mul tiple assays for the same gene should result in the same Cg values validating the true expression level of that gene under the specific experimental conditions 7 2 4 Baseline Baseline set incorrectly The baseline is a critical component for determining accurate Cg during qPCR data analysis If set wrong amplification results can appear reduced or delayed The baseline should be wide enough to eliminate background that occurs in early cycles of amplification bu
72. ions it is still very important to assess the quality of the experiment particularly to determine its sensitivity A target may appear to be absent when it is actu ally present in a low amount because it is not detected due to the experimental setup 58 AXCXIDI INTEGRATED DNA TECHNOLOGIES 7 PrimeTime qPCR Assay Troubleshooting As qPCR is a complex multifaceted process suboptimal amplification may be observed for a number of reasons Troubleshooting or generation of additional data may be re quired to achieve optimal results Stylized examples of the types of problematic qPCR data that can be encountered are shown following Match your data with one of these and refer to the indicated section to learn what causes such curves and how to remedy them Good qPCR curves should look like these Problem gPCR Curves Your curves look like this paa You likely have too much template See Section 7 4 4 59 aPCR Application Guide Your curves look like this m m There is no amplification See Section 7 1 Missing reaction component Several factors can contribute to this Sample degradation Incorrectly assigned dye detector No target in sample Ca y Poor assay design Your replicates look like this Instead of this Inconsistent replicates See Section 7 6 Several factors can contribute to this Pipetting errors Thermal calibration of thermal cycler Low target copy number Inappropriate cycling condi
73. k Try reposi tioning the samples that are giving the delayed signal into different wells to see if results improve If so it suggests that the block is not heating uniformly and the real time PCR instrument requires a temperature calibration Thermal cyclers usually require calibration for temperature consistency once every 6 months Most instruments have a built in test run or self check protocol that allows the instrument to recalibrate itself if determined necessary You can also use temperature sensitive dyes Life Technologies compatible with most instruments to calibrate the thermal cycler Determine whether your instrument has a built in temperature calibra tion self check protocol New light source needed f the instrument has been recalibrated for the dyes used but samples continue to provide inconsistent results when tested in different wells it is pos sible that the instrument needs a new light source Refer to your instrument manual for instructions on how to replace a light source bulb 7 6 4 RNA Sample Quality cDNA replicates yielding variable Cg values It is possible that during RNA sample prepa ration the quality of some samples was compromised e g by degradation which is reflected in the cDNA product Figure 21 shows how degraded template can affect qPCR amplification Poor template quality can be due to varioius factors including the RNA isolation method poor reverse transcription and improper storage and hand
74. ling Check sample quality by assessing the RNA integrity with a system such as Experion BioRad or 2100 Bioanalyzer Agilent or by examining a small amount on a gel see Section 2 3 Use TE buffer to resuspend the RNA sample Check the RT reagents for contamination or expiration 28 aPCR Application Guide Initial dilutions of standard curve not producing expected Cy intervals Contaminants may be present in the sample These can originate from the host tissue or cell They can also be present in enzymes used in the RT reaction or qPCR components used to isolate the RNA or other reagents added in the process Such contaminants can inhibit the am plification of the sample Inhibition by contaminants is often more pronounced in the least diluted standards when the contaminants are still fairly concentrated To check for inhibitors include a serial dilution of your sample in an endogenous con trol assay The highest concentration of template contains the highest concentration of inhibitor which causes a delayed Ca In contrast a lower concentration contains less inhibitor resulting in an earlier Cg and a change in the slope No Treatment 104 Oligo dT Primed m 8 ng cDNA E 0 8 ng cDNA E 0 08 ng cDNA Average Cq Val NNNNNNNNNNWWWWWW CENWEUC IRON OEM OOooooooooooooooo w a ww Nw oo E 8ng cDNA E 0 8 ng cDNA 0 204 34 0 V 32 10 S 30 M E g g 1 1 T T T T Avera
75. m under the Support tab and can also be found on the IDT YouTube channel at www youtube com idtdnabio The webinar Technical Tips for qPCR Assay Design and Setup focuses on 5 nuclease assay design and experimental setup and provides guidance on design parameters such as Tm GC content amplicon size and location of primers and probe Another useful webinar Using Free Online Tools for Oligonucleotide Analysis and Design discusses the use of the SciTools programs described in Section 8 2 1 This webinar cov ers the use of the SciTools programs specific for designing PCR and RI qPCR experi ments It also discusses use of the OligoAnalyzer tool for basic oligonucleotide analysis including sequence analysis for thermodynamic properties of dimers and hairpins 84 AXCXIDI INTEGRATED DNA TECHNOLOGIES 8 2 4 DECODED Newsletter The quarterly IDT DECODED newsletter is a free resource available in print and online in HTML and PDF formats It includes easily accessible articles that cover a variety of help ful scientific topics including articles providing tips and information on many aspects of RT qPCR Past issues are available online at www idtdna com Register for an electronic or print subscription at www idtdna com decoded 8 3 Other Resources 8 3 1 BLAST Analysis NCBI s Basic Local Alignment Search Tool BLAST is an incredibly powerful tool that can be used to efficiently query the massive Genbank database to find regions o
76. me the mRNA of interest yield the least complex cDNA mixture and avoid reagent depletion The main disadvan tage to their use is that the cDNA produced cannot be used for assaying other genes 21 aPCR Application Guide Oligo dT primers will ensure that mRNA containing poly A tails are reverse transcribed These primers are more commonly used when trying to limit the amount of ribosomal RNA being copied or when the qPCR assays are designed to target the 3 end of the RNA If the mRNA is long the 5 end of the message may be under represented 3 3 Replicates and Controls Variation can be easily introduced at this step in the process so it is very important that all samples are treated the same including the input amount of RNA the priming strategy the enzyme type the volume of the reaction the temperature used and the reaction time 1 For accurate analysis of RT qPCR results each experiment needs to be set up with multiple replicates and controls See Section 4 2 6 Figure 14 for a schematic outlining assay setup Replicates For each experimental and control sample to be compared it is highly rec ommended that at least three biological replicates are used The number of technical replicates performed is dependent on the steps taken to minimize errors due to poor pipetting or uncalibrated equipment and on the precision required No RT Control For every reverse transcription reaction it is important to incorporate a no RT
77. mplification Plot Amplification Plot Amplification Plot 1 000 Ed Correct Probe Concentration 1 000 1 000 E 2 1 000 E 1 at A A A A A O 1 000 E31 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 Detector FAM TAMRA Plot Rn vs Cycle Detector FAM TAMRA Plot ARn vs Cycle y Threshold 0 09881737 Figure 22 Low Signal Due to Incorrect Probe Concentration Rn Linear View 75 aPCR Application Guide 7 7 2 Primer and Probe Integrity Inadequate probe purification High background can also result from poor purifica tion of the probe IDT performs HPLC mass spectrometry and capillary electrophoresis on each probe synthesized to ensure probe integrity and purity These OC data are all posted on the customer s web account at no extra charge Probe degraded prior to cycling Degradation of the probe at the start of the experi ment will be indicated by extremely high background fluorescence that does not change with cycling This is especially likely if the background fluorescence has changed since a previous run with the same probe If the probe has degraded you will need to use a new aliquot if available or a newly synthesized probe IDT recommends that the probe be resuspended in TE buffer 10 mM Tris pH 7 5 8 0 0 1 mM EDTA for storage Water is a poor choice of solvent as it can be acidic and cause depurination and strand scission Always store probes in aliquots at
78. multiplex assay Inability to detect the target with the least expression A more abundant target may amplify more efficiently than a less abundant target and compromise the entire reaction This is of particular concern in later cycles when the dNTPs and Tag polymerase are limit ing Limit the primers for the highest expressing targets to a 1 to 1 primer to probe ratio while increasing the primer to probe ratio of the other targets if necessary Also use a FAM labeled probe for the target with the least expression FAM is the brightest emitting dye and will ensure maximum sensitivity Use double the amounts of dNTPs and enzyme in the master mix See Section 4 2 4 for more information on setting up multiplex assays 80 AXCXIDI INTEGRATED DNA TECHNOLOGIES 7 10 Other Observations 7 10 1 Rising Baseline Make sure the baseline is set correctly refer to sections 6 2 and 7 2 4 If necessary set the baseline manually It is also possible that there is primer probe interaction or the primers are forming primer dimers Make sure to run a no template control Evaluate the cross reactivity of each assay component The OligoAnalyzer Tool is ideal for this purpose See Section 8 2 2 for more information 7 10 2 Variations in C of normalizer gene The data gathered from normalization will only be as good as the control used Figure 24 Make sure that the control has been verified as appropriate for your sample before you use it as a normalizer
79. n analysis of the RNA integrity value A ratio of 1 8 indicates the RNA is of good quality Lower ratios could be due to organic compound contamination Turbidity or low pH can also lead to calcula tion errors To correct for the effects of turbidity when estimating RNA quality at neutral OH readings at 320 nm should be subtracted from readings at 240 260 and 280 nm Al ternative methods for determining quality include gel electrophoresis microfluidics based rRNA analysis or a reference gene target gene 3 5 integrity assay 1 15 2 4 Avoid RNases DNases RNases and DNases are nucleases that can quickly degrade samples and oligonucleotide primers and probes They are ubiquitous and can be difficult to eliminate Therefore it is very important to take precautions to ensure that samples are protected from degrada tion by these nucleases Follow clean PCR guidelines see next page to prevent con tamination and test the samples using nuclease detection reagents such as RNaseAlert and DNaseAlert Kits IDT If you do find contamination in your samples be sure to replace all reagents and stock buffers and thoroughly clean the PCR preparative areas RNase inhibitors can be added to block the action of some ribonucleases and DNases can be inactivated by heat treatment Related Products at IDT RNaseAlert and DNaseAlert Kits These reagents are fluorescence quenched oli gonucleotide probes that emit a fluorescent signal only after nu
80. n if it has previously been reported in the literature or is commercially available to verify that the assay is working according to standards under the conditions you will be using 5 1 Specificity Analysis It is important to validate assay specificity to ensure that only the sequence of interest is detected Determine specificity using one of 3 different characteristics the melt curve size or Sequence of a PCR product 5 1 1 Melt Curves When performing assays that use intercalating dyes as the detection chemistry you can use melt curves to analyze the specificity of the assay Melt curves are characteristic for the PCR products generated Therefore if multiple PCR products are formed e g as a result of nonspecific amplification multiple melt peaks will likely be present Specific amplification is typically characterized by a unique melt peak although unique products may show multiple peaks if the sequence contains domains that melt out at different temperatures Predict whether this will be the case using a software package such as uMelt available at http dna utah edu umelt umelt html 5 1 2 Amplicon Size Analysis By performing size analysis of a PCR product on agarose gels or microfluidic devices you can detect any nonspecific products that have formed It is possible for two dif ferent products to be of the same size e g in case of co amplification of homologous sequences pseudogenes members of same gene family etc
81. nes are compact Trends Genet 19 362 365 Arezi B Xing W et al 2003 Amplification efficiency of thermostable DNA polymerases Anal Biochem 321 2 226 235 Hellemans J Mortier G et al 2007 qBase relative quantification framework and software for management and automated analysis of real time quantitative PCR data Genome Biol 8 2 R19 Willems E Leyns L and Vandesompele J 2008 Standardization of real time PCR gene expres sion data from independent biological replicates Anal Biochem 379 1 127 129 Pfaffl MW 2006 Relative quantification In Dorak T editor Real time PCR La Jolla CA Inter national University Line p 63 82 Huggett J Dheda K et al 2005 Real time RT PCR normalisation strategies and consider ations Genes Immun 6 4 279 284 Vandesompele J De Preter K et al 2002 Accurate normalization of real time quantitative RT PCR data by geometric averaging of multiple internal control genes Genome Biol 3 7 RE SEARCHO0034 Livak KJ and Schmittgen TD 2001 Analysis of relative gene expression data using real time quantitative PCR and the 2 Delta Delta C T Method Methods 25 4 402 408 aPCR Application Guide 10 Notice of Limited Licenses NOTICE TO PURCHASER LIMITED LICENSES IDT manufactures and sells PrimeTime qPCR Probes for research purposes only Texas Red is a registered trademark of Molecular Probes Life Technologies and is licensed for research use under paten
82. ng temperature 4 2 2d Calculating Melting Temperature Tm During the annealing step of PCR primers and probes hybridize to targets forming short duplexes The stability of these duplexes is described by the melting temperature the temperature at which an oligonucleotide duplex is 5096 single stranded and 5096 dou ble stranded Figure 11 Melting temperature is a key design parameter Inaccurate Tm predictions increase the probability of failed assay design IDT provides several free software tools SciTools ap plications on the website that can predict Tm values from oligonucleotide sequences and reaction composition It is often mistakenly believed that Tm is solely a property of the oligonucleotide sequence and independent of experimental conditions Melt ing temperature depends on oligonucleotide sequence oligonucleotide concentration and cations present in the buffer specifically monovalent Na and divalent Mg salt concentrations Figure 12 For this reason the melting temperature for specific ex perimental conditions should be calculated using IDT SciTools design tools see Section 8 2 1 for information on which SciTools application best fits your needs 30 AXCXIDI INTEGRATED DNA TECHNOLOGIES Figure 11 Melting Profiles for Primers of Various Lengths Reactions were performed using a PCR buffer containing 1 mM Mg 50 mM KCI 10 mM Tris GC content for all prim ers was 50 of duplex 15 bas
83. nsity The presence of an internal quencher also makes the use of longer probes possible See Section 4 1 3 for more information 76 AXCXIDI INTEGRATED DNA TECHNOLOGIES Probe degradation A degraded probe should exhibit constant fluorescence To check probe integrity perform a signal to noise ratio STNR assay Dilute an aliquot of the probe to a final concentration of 0 25 uM Add 1U micrococcal nuclease and digest the sample at 37 C Measure the increase in fluorescence over a background reaction of probe plus buffer without micrococcal nuclease An intact probe should exhibit in creased fluorescence 7 7 3 Instrument Fluorescent contaminants It is possible that the thermal block contains fluorescent contaminants resulting in high background and decreased signal Run a background or water plate to confirm the background is still within specification Refer to the instru ment manual or contact a service organization 7 8 Passive Reference Problems only applies to instruments that use ROX dye The signal of the passive reference should be significantly higher than the background signal of the instrument Check your master mix to see if it has the correct passive refer ence concentration high or low ROX for the instrument that you are using The use of high or low ROX will depend on the instrument refer to the instrument manual for the appropriate concentration 7 8 1 Lower than expected amplification curves high ROX I
84. nt Replicates s ea ur ae riea ee 72 1 6 1 Reaction SetU D vus eoo oe oec he 9 a a ia A fa 7 0 2 gt ao ETA 725 7 6 3 Instrument Calibration uuu a uod x Ro v RR hne cxx XO Oy Gwe 3egod amp Re 13 TOA RNA Sampe QUAI e s on eso iria UR e 3e Ros de A dod pe cm 8 3 7 7 High or Variable Background 75 LACAN Uan aa a eh a a A o 75 7 7 2 Primer and Probe Integrity ss ux baw 4 m a oo HRS 76 FUA AS ee ee Oe Bk 77 7 8 Passive Reference Problems only applies to instruments that use ROX dye 77 7 8 1 Lower than expected amplification curves high ROX o o o Li 7 8 2 Higher than expected or noisy amplification curves low ROX 7 7 8 3 Amplification curve drops off and has an atypicalshape 78 7 8 4 When used with ROX the TAMRA signal is diminished 78 7 9 Multiplexing PIODIGING sa 6 40 eee oe ow ae 30x Owe SO eR DE 80 IONES DOSES aue acu dr qe anar PP dex E SOR SENG Seded OR 81 IO A RISTO BASES m 81 7 10 2 Variations in Cg of normalizer gene 2 ees 81 8 RT qPCR Additional Resources o 82 MN o ao A EIN 82 8 2 IDT Resources In Print ana Online sess eae dieu X X ria de a ORs 82 924 Sc oo PCR Assay Design TOOIS p easier do pow Ge ange ye des 82 8 2 1a Predesigned qPCR Assays Tool 2 a 83 8 2 15 RealTime PCR Assay Tool 2 ees 83 8 2 10 PrimerQuest Design TOO a aou v de gt seri karra a pe e 83 8 2 2 Additional SciTools Software
85. nterest treat samples that may con tain genomic DNA contamination with DNase prior to cDNA synthesis see Section 2 1 When possible design primers probes or amplicons to span an exon exon junction to avoid amplification of genomic DNA see Section 4 2 1 1 aPCR Application Guide 7 4 4 Template Concentration Addition of too much template The template concentration is too high if Cg values ear lier than 15 are obtained The addition of too much template may cause miscalculation of the baseline factor and affect the shape of the curve Dilute the template as necessary to ensure that Cg values are greater than 15 7 5 Noisy Data Addition oftoo much probe High probe concentration is usually due to errors in dilution or reaction setup Refer to the protocol to verify that probe was added and at the appro priate concentration See Section 4 3 2 for a protocol Also ensure that calculations for the dilution of probe are correct and that the probe was diluted appropriately Less likely is that the recommended primer and probe concentrations are not ideal for your sample This can be tested by decreasing the concentration of probe stepwise by 25 nM Insufficient reference dye ROX degradation or low ROX concentration can cause the ARn to be high and noisy as the instrument tries to normalize data using a weak inconsistent ROX signal See 7 8 2 for more details 7 6 Inconsistent Replicates As a general guideline replicates should n
86. of fluorescent intensity is a function of both the intrin sic properties of the compound such as quantum efficiency and molar absorptivity and the incident radiant power Therefore using a dye of low fluorescent capability on an instrument that has not been calibrated for that dye may result in reduced height of the amplification curve 69 aPCR Application Guide 7 3 Poor Efficiency 7 3 1 Design Specificity Cg higher than expected value This observation likely indicates low primer efficiency There may be mismatches between the target and primer probe sequences or non optimal PCR conditions Perform a BLAST search to confirm the specificity of the target and assay sequences See Section 8 3 1 for more information about this free NCBI tool Run a gel to see if the correct size of product is being amplified If you are not using a predesigned assay from IDT check that your primer and probe do not span a SNP site Note that all PrimeTime qPCR Assays are designed with up to date sequence informa tion specifically to avoid assay design over SNP sites thus preventing this problem 7 3 2 Reaction Setup Efficiency of the reaction has decreased over time Rerun an assay that has worked previ ously to see if the problem is assay specific If so assay degradation may have occurred Reorder the assay if necessary If the decrease in efficiency is not assay specific it is possible that the master mix was improperly stored or that the template h
87. on 4 1 000 E 1 1 000 E 2 Cycle Detector E FAM Plat aRn vs Cycle Threshoict 0 1789872 33 aPCR Application Guide 6 4 Determining Gene Expression Changes Gene expression is quantified by the number of cycles required for fluorescence sig nal to reach the threshold During amplification the fluorescence from a more highly expressed gene will reach the threshold at an earlier cycle than a gene with a lower level of expression To analyze the amount of PCR product investigators compare either absolute levels of RNA copies for each sample or relative levels of RNA to an unchanged control 8 6 4 1 Absolute Quantification Absolute quantification requires generating an absolute standard curve for the gene of interest and calculating the number of gene copies in an unknown amount of sample from a comparison with the standard curve The standard curve can be created using a known amount of DNA linearized plasmid PCR amplicon or oligonucleotide The un known test sample amount can then be interpolated from the standard curve calcula tion The reliability of this method is dependent on identical amplification efficiencies of the known and test samples 28 and on the accuracy with which the standard samples are quantified 6 4 2 Relative Quantification Relative quantification is the more commonly used method of analysis and is expressed as the fold difference in gene expression between test and control
88. ot vary by more than 0 5Cg however this can be more stringent based on he differences in the samples being analyzed 7 6 1 Reaction Setup Poorly mixed reaction This is a common mistake that can result in a large spread in the replicates and irregular spacing between dilutions of the standard Be sure to gently mix the sample after all components are added Master mix not used Every single sample should be treated in the same manner in order to achieve reliable results For that reason it is very important to use a master mix to en sure that every sample receives the same amount of each of the reaction components See Section 4 3 2a for more information on master mixes Pipetting errors Ensure that your pipettes are properly calibrated and that the seals are in good repair Use pipette tips once only and ensure other good pipetting techniques 72 AXCXIDI INTEGRATED DNA TECHNOLOGIES 6 2 Reaction Parameters Activation step not long enough It is possible that the PCR activation step was not long enough for the enzyme used Some hot start enzymes require longer activation at 95 C than others Check the requirements for the enzyme you are using 7 6 3 Instrument Calibration Temperature calibration needed f for example replicates are inconsistent or the Cy dif ference between successive 10X dilutions is not 3 32 the instrument may require tem perature calibration This is likely if results are inconsistent across the bloc
89. p For information concerning availability of additional licenses to practice the patented methodologies please contact Amersham Biosci ences Corp Business Licensing Manager Amersham Place Little Chalfont Bucks HP79NA UK EvaGreen is a registered trademark of Biotium Inc TYE MAX TEX 613 OligoAnalyzer PrimerQuest and SciTools are trademarks of Inte grated DNA Technologies FAM HEX ROX TAMRA and TET are trademarks of Life Technologies Corporation LightCycler Dyes are a registered trademark of a member of the Roche group and are sold under license from Roche Molecular Diagnostics GmbH Molecular Beacons are sold under license from the Public Health Research Institute only for use in a purchaser s research and development activities Scorpions is a registered trademark of DxS Limited LTD SYBR Green is a registered trademark of Molecular Probes Inc TaqMan is a registered trademark of Roche Molecular Systems that is licensed exclusively to Applied Biosystems Inc for use in certain non diagnostics fields 88 AXCXIDI INTEGRATED DNA TECHNOLOGIES TYE Dyes are sold under license from Thermo Fisher Scientific Milwaukee LLC The trademarks mentioned herein are the property of Integrated DNA Technologies or their re spective owners 69 aPCR Application Guide 11 Index A Amplicons 30 Length 30 Melting temperature Tm 30 Amplification 7 Efficiency 47 49 58 Exponenti
90. program such as qbasePUS from Biogazelle see Section 8 3 2 for more information 6 1 Rn ARn and RFUs The reporter and the reference dyes if used with your instrument will have particular fluorescence emission intensities The normalized reporter signal referred to as Rn on some instruments is the ratio of the fluorescence emission intensity of the reporter dye to that of the reference dye A reference dye ROX is often used to normalize for any non PCR related fluctuations in the reaction This reference dye is not required for all instruments but is a helpful tool for troubleshooting Instruments that do not use a ref erence dye often refer to the fluorescent signal as relative fluorescence units RFUs The reporter signal Rn minus the baseline fluorescence seen in early PCR cycles is referred to as baselined relative fluorescence units on some instruments and ARn on others ARn or baselined RFUs plotted against cycle number generates the amplification curves and the Cg value 6 2 Setting the Baseline The baseline is the fluorescence present in the initial cycles of PCR prior to a detectable increase in signal resulting from accumulation of the amplicon The baseline should be set in the normalized linear view ARn vs cycle and should be wide enough to eliminate the background found in early cycles of amplification However it should not overlap with the area in which the amplification signal begins to rise above background Figure
91. protein large PO RPS18 ribosomal protein S18 RPS27A ribosomal protein S27a SFRS9 splicing factor arginine serine rich 9 TBP TATA box binding protein TFRC transferrin receptor UBC ubiquitin C YWHAZ 3 monooxygenase tryptophan 5 monooxy genase activation protein zeta polypeptide Table 4 Genes Commonly Used for Normalization aPCR Application Guide Related Products at IDT Both Ultramer Oligonucleotides and gBlocks Gene Fragments serve as excellent controls and importantly can serve as standards of known concentration For more information and to order Ultramer Oligonucleotides and gBlocks Gene Fragments visit the IDT website at www idtdna com Ultramer Oligonucleotides IDT synthesis systems and chemistries allow high fidelity synthesis of very long oligonucleotides up to 200 bases Ultramer Oligo nucleotides are suitable for demanding applications such as cloning shRNA muta genesis and gene construction greatly reducing effort and time by having entire target fragments synthesized by IDT gBlocks Gene Fragments IDT offers double stranded DNA fragments up to 500 bp in size gBlocks fragments are constructed using Ultramer Oligonucleotides and are sequence verified 4 3 PrimeTime gPCR Assay Protocol This protocol is intended for use with IDT PrimeTime qPCR Assays Concentrations and volumes may vary for other products 4 3 1 Resuspension Protocol 1 Centrifuge PrimeTime qPCR Assay tubes at 750 x g
92. r Promega 4 Incubate at 42 C for 2 min 5 Add 0 5 uL Superscript Il Life Technologies 6 Incubate at 42 44 C for 1 hr 7 Incubate at 70 C for 15 min For random hexamers incubate at 25 C for 15 min to allow some extension and then increase temperature to 42 44 C 23 aPCR Application Guide 4 Real Time qPCR Design and Protocols To achieve reliable interpretable results from qPCR the following important factors must be considered Primer and probe design are crucial to the success of the experiment The real time PCR instrument will dictate certain parameters of the experiment im portantly some instruments are not compatible with some fluorescent dyes Table 2 see page 35 lists several dyes and associated instrument compatibility However this list is limited and may be subject to change Check the manual for your particu lar instrument to verify compatible dyes and correct cycling conditions If you are running a multiplex experiment additional considerations will need to be incorporated particularly in the assay design and choice of dyes As a final step before the reaction is set up determine the controls that you will run and be sure to calculate those extra reactions into your total number of reactions Include both positive and negative controls This section includes recommendations for design as well as protocols for resuspension and reaction setup Always use RNase and DNase free reagent
93. r each dye to be calibrated Follow instructions in the instrument software menu to perform calibration for each dye Review data to identify variations in dye spectra Refer to the instrument manufacturer for specific instructions 4 2 6 Replicates and Controls For accurate analysis of qPCR results each experiment needs to be set up with multiple replicates and controls Figure 14 4 2 6a Replicates qPCR experiments use two different types of replicates Technical replicates repeated RT or qPCR reactions are used to compensate for technical noise and to increase the precision of the qPCR results These repeated measures should not be used for statistical 36 AXCXIDI INTEGRATED DNA TECHNOLOGIES testing of biological hypotheses Biological replicates repeated cell cultures or different individuals or specimens are required to draw biologically relevant conclusions from your experiments The number of replicates depends on the specific needs of the experiment For each experimental and control sample to be compared at least 3 biological and 3 technical replicates are necessary to minimize errors in measured gene expression due to pipet ting And at least 3 biological replicates are required if you want to draw statistical con ci usions See Figure 14 for an example However if you are interested in small expression differences or require higher confi dence e g for diagnostics it is useful to include more replic
94. r which the concentration of the different genes is not known Such relative standard curves are typically used for assay performance assessment and quality control of the experiment Creating a standard curve requires setting up additional reactions however the data obtained can be very important for determining the quality of the PCR Researchers ex perienced in qPCR often consider it valid to assess assay performance using a standard curve only once and then never use a standard curve again particularly in a research setting where many different assays are used 5 2 2 Range of Dilution A standard curve across multiple logio units is needed Figure 16 The concentrations should span a minimum of 4 logio of magnitude but preferably 5 6 logio The concen trations of the test unknowns should fall within the range of concentrations used for the standard curve without the need to extrapolate The PCR efficiency is close to 10096 when the slope of the amplification curve is close to 3 32 See Section 5 3 for more information on PCR efficiency 47 aPCR Application Guide 5 2 3 Template There are several methods available for preparing material for a standard curve A PCR product can be amplified from the target of interest cloned or purified and then seri ally diluted Alternatively to avoid the additional steps required for cloning a PCR prod uct long oligonucleotides Ultramer Oligonucleotides or gBlocks Gene Fragments
95. rnal ZEN quencher While the distance between fluorophore 3 Quencher Distance from Dye 20 30 bp AA o f 3 Quencher Distance from Dye 20 30 bp i ZEN Distance from Dye 9 bp EL p Qa Figure 9 Location of ZEN Quencher 25 and quencher in traditional probes is 20 30 bases the internal ZEN quencher decreases this distance to only 9 bases Figure 9 This shortened distance particularly when combined with the traditional 3 end quencher leads to extremely efficient quenching with significantly less background Figure 10 and enables the use of much longer probes for designs falling within AT rich target re aPCR Application Guide gions In addition to the greatly decreased background the double quenched probes allow users to experience increased sensitivity and greater precision in their qPCR ex periments Double quenched probes are available as PrimeTime Custom qPCR Assays PrimeTime Predesigned qPCR Assays or sequences specified by the customer Rn Amplification Curves delta Rn Amplification Curves 0 5 ng cDNA per Reaction 0 5 ng cDNA per Reaction 4 ex 5 FAM ZEN 3 IBFQ ex 5 FAM 3 BHO 1 3 e5 FAM 3 Eclipse 5 FAM 3 IBFQ 5 FAM 3 TAMRA 5 FAM ZEN 3 IBFQ 5 FAM 3 BHO 1 5 FAM 3 Eclipse 5 FAM 3 IBFQ 1 5 FAM 3 TAMRA Rn normalized fluorescence delta Rn normalized fluorescence 0 10 20 30 40 50 0 10 20 30 40 50 Cy
96. roduct creating a 1 1 ratio between the flu orescence emission of a cleaved probe and recognition of one amplicon molecule aPCR Application Guide Related Products at IDT IDT offers PrimeTime qPCR products for the 5 nuclease assay PrimeTime qPCR As says consist of a forward primer a reverse primer and a labeled probe all deliv ered in a single tube The PrimeTime qPCR probe is a non extendable oligonucle otide that is labeled with a fluorescent reporter and a quencher dye PrimeTime Predesigned qPCR Assays are available in three different scales and five different dye quencher combinations for human mouse and rat transcriptomes PrimeTime Predesigned qPCR Assays are prepared when ordered using up to date sequence information from NCBI and are guaranteed to be at least 9096 efficient when used with a commercially available master mix and measured over 4 orders of magnitude IDT offers PrimeTime qPCR Primers that are ideal for SYBR Green or other interca lating dye assays where no probe is needed These predesigned primer pairs are identical to those in PrimeTime Predesigned qPCR Assays For increased sensitivity and decreased background IDT also offers assays and probes with the internal ZEN Quencher Double Quenched Probes available with FAM HEX and TET dyes Cus tom primers molecular beacons and hybridization FRET probes can all be obtained mon DOT In addition IDT offers SciTools Design Tools a suite of free design and
97. rs e g 94 096 1 2 and propa gates this uncertainty in the final error bars for the results see Section 8 3 2 for more information about Biogazelle software tools The errors in the estimated PCR efficiency are generally smaller when using more dilution points or a wider dilution range 5 4 Limit of Detection LOD and Limit of Quantification LOQ The limit of detection LOD is the lowest concentration at which 9596 of the positive samples are detected 1 The LOD indicates the concentration at which signal is lost which is also where the measurements are no longer valid To determine the LOD exam ine the standard curve to find the most dilute sample that is still detectable in each case and with variance less than 1 Cg see Section 6 3 for an explanation of Ca Ideally the no template control NTC reaction should not register a Cg value However if a Cg value is measured for the NTC reaction the Cg of the most dilute test solution must be several Cq values lower than the Cg of the NTC to ensure reliability a difference of 5 cycles would correspond to a contamination or background signal of 396 which is below the precision of qPCR Only Cg values that fall within the LOD are reliable It is very important not to attempt to extrapolate Cg values that fall outside the lowest value The limit of quantification LOQ is the lowest amount of target that can be quantified with accuracy and reproducibility so that conclusions based on t
98. s Suboptimal amplification can be improved by adjusting the reverse transcription reac tion and or the PCR cycling conditions Nonoptimal reverse transcription conditions Check the following parameters Length Change the length of the reverse transcription step in 5 minute increments up to a maximum of 60 minutes Temperature The reaction should be set up on ice so that cDNA synthesis does not begin prematurely Change the temperature of the reverse transcription reaction in 5 C increments up to a maximum as determined by the enzyme and the type of primer you are using Nonoptimal PCR conditions Check the following parameters Annealing and extension times Figure 19 shows the effect of different annealing extension times on qPCR amplification Annealing or extension steps that are too short can result in little or no amplification When the annealing step is too short amplification can be inconsistent or decreased or may not occur at all As a general 64 XIDT INTEGRATED DNA TECHNOLOGIES recommendation adjust the time in 15 second increments up to a maximum of 1 minute for amplicons that are 250 bp or shorter when using standard cycling condi tions for fast cycling master mixes use 3 second increments Refer to the user guide for your master mix for more specific recommendations a Amplification Plot Amplification Plot Amplification Plot Amplification PI Amplification Plot Amplification Plot
99. s check their expiration dates and verify their concentrations Although many of these considerations can be used for any 5 nuclease assay product the protocol in Section 4 3 is intended for use with PrimeTime qPCR Assays from IDT 4 1 PrimeTime gPCR Assays and Associated Products PrimeTime qPCR Assays consist of 2 primers and a hydrolysis probe delivered in a single tube These assays simplify qPCR optimization by allowing for selection of dyes addi tion of a second quencher see Section 4 1 3 choice of primer to probe ratios and adjustment of qPCR design parameters PrimeTime qPCR Assays selected from the IDT Predesigned Assay collection or generated with the RealTime PCR Design Tool for Custom Assays are guaranteed to provide assay efficiency gt 90 when used with a com mercially available master mix and measured over a minimum of 4 orders of magnitude PrimeTime Custom and Predesigned qPCR Assays are offered in three different sizes to allow researchers flexibility for a range of experimental needs from screening many genes to testing the same endogenous controls over many experiments 24 AXCXIDI INTEGRATED DNA TECHNOLOGIES 4 1 1 PrimeTime Custom gPCR Assays PrimeTime qPCR Assays can be custom designed using the sophisticated free IDT qPCR primer and probe RealTime PCR Design Tool found in the SciTools section of the IDT web site www idtdna com Simply enter the RefSeq numbers or paste in a sequence for de sign Th
100. s over single reaction PCR including requiring a lower amount of starting material increased throughput lowered reagent costs and less sample handling However the experimental design for multiplexing is more complicated because the amplification of each target can affect others in the same reaction Therefore careful consideration of design and optimization of the reactions is critical In order to incorporate all necessary parameters we strongly recommend using a design tool for primers and probes Suitable tools can be found in the SciTools applications section of the IDT website See the Section 8 2 1 for more information 1 Ensure that the primers and probe sets are not complementary to each other Use BLAST to analyze the sequences and ensure they are non complementary For help with using the BLAST tool see Tips for Using BLAST to Locate PCR Primers in the IDT DECODED 1 1 April 2011 newsletter 2 Each target must be identified by a separate reporter dye Select dyes with little or no overlap in their emission spectra Table 3 Some instruments are compatible with only certain dyes read Section 4 2 3 so check the documentation for your instrument to ensure the dyes are compatible As a general rule it is a good idea to select FAM for any low copy transcripts because it has a strong signal Lower signal fluorophores can then be used for the more abundant transcripts 3 Minimize signal cross talk by using high quenching probes su
101. samples for a given gene Importantly relative quantification cannot be easily used to compare expression levels between genes due to the assay dependent relationship between Cg value and input amount To normalize input quantities expression of the target gene of interest is typically compared to one of the following an endogenous control reference gene an exogenous control a reference gene index or a target gene index 28 Relative quanti fication measures the difference between the Cg values 28 The equation used de pends on the similarity or differences in the efficiencies of the reactions and the number of reference genes being used 26 54 AXCXIDI INTEGRATED DNA TECHNOLOGIES 6 4 2a Normalization Normalization corrects for inequalities in DNA concentrations resulting from variations during sample preparation to enable comparison across samples Typically normaliza tion uses one or more reference genes as an internal control for this comparison How ever samples may also be normalized to sample size total RNA or to an artificial mol ecule included in the reaction 29 Samples that are being compared must have been subjected to identical sampling isolation reverse transcription and qPCR conditions In addition the results obtained depend on the quality of the normalizer chosen so veri fying a representative normalizer that has identical expression across all samples tested is vital for the accuracy of the re
102. se will depend on the RNA source and experimental conditions of the sample you will be testing It is best practice to screen multiple genes under the experimen tal conditions employed to determine which expression levels fluctuate the least Programs such as geNorm can be used to evaluate the performance of various normalizers The most commonly used normalizers are listed in Table 4 24 Alterna tively review the literature for the genes tested on samples with conditions similar to yours If you choose a normalizing gene from the literature be sure to perform a reaction to verify that the gene expression levels do not fluctuate across samples before you use it as a control 38 AXCXIDI INTEGRATED DNA TECHNOLOGIES GENE ID Description 185 185 ribosomal RNA ACTB actin beta ALDOA aldolase A fructose bisphosphate ARHGDIA Rho GDP dissociation inhibitor GDI alpha B2M beta 2 microglobulin GAPDH glyceraldehyde 3 phosphate dehydrogenase GUSB glucuronidase beta HMBS hydroxymethylbilane synthase HPRT1 hypoxanthine phosphoribosyltransferase 1 HSPCB heat shock 90kDa protein 1 beta IPO8 importin 8 LDHA lactate dehydrogenase A NONO non POU domain containing octamer binding PGK1 phosphoglycerate kinase 1 PGK1 phosphoglycerate kinase 1 POLR2A polymerase RNA II DNA directed polypeptide A PPIA peptidylprolyl isomerase A cyclophilin A RPL11 ribosomal protein L11 RPL19 ribosomal protein L19 RPL32 ribosomal protein L32 RPLPO ribosomal
103. solutions DNA dissolved in distilled water often has a final pH 5 0 and is at risk of depurination IDTE is guaranteed to be nuclease free Each lot is tested using our RNaseAlert and DNaseAlert Kits to document the absence of nuclease activity For more information and to order IDTE buffer visit the IDT website at www idtdna com 4 3 1a Avoiding Probe Degradation In order to avoid probe degradation resuspend primers and probes in TE buffer 10 mM Tris 0 1 mM EDTA pH 8 0 rather than water as TE buffer will maintain a constant pH Store probes away from light Oligonucleotides should be distributed into aliquots for immediate use or long term storage Storage in aliquots will help minimize the risk of contamination of the stock solutions 4 aPCR Application Guide 4 3 2 Assay Protocol Most qPCRs are performed in 20 uL 10 uL or 5 uL reaction volumes It is very important that the cDNA and reaction volumes remain constant across all samples that need to be compared IDT recommends that you perform a minimum of triplicate reactions for each sample Remember to include control samples when calculating the number of reactions you need for each assay 1 Inasterile 1 5 mL microcentrifuge tube pipette the assay master mix and water in the volumes listed based on the number of assays you need to run Table 6 Cap the tube and vortex to mix Centrifuge the tube at 750 x g for 10 sec 2 Depending on the number and total volume of reac
104. sults e Normalizing to a reference gene When normalizing to a reference gene it is very important that the reference gene is experimentally validated to ensure that it is an accurate measure against which to com pare all other sample variations Ideally more than one reference gene should be tested A pilot study can be conducted to select the best set of reference genes out of a series of candidates Analysis of reference gene stability can be performed with tools such as geNorm 30 and qbase 5 26 The reference genes should have stable mRNA expres sion and the amount of reference gene mRNA should be strongly correlated with the total amounts of mRNA in the samples 1 When using this method it is critical that the reference genes used do not vary with experimental conditions Normalized data is reported as a ratio of the mRNA concentration of the gene of interest to the mRNA con centration of the reference gene 1 This can be calculated by a comparative Cg method or a standard curve method e No efficiency correction This method is based on the assumption that all the reactions occur with 10096 efficien Cy so that there is a doubling of the target DNA with each cycle of PCR Such conditions are unlikely with multiple assays and therefore we do not recommend this method Small differences in PCR efficiencies between targets and reference genes can lead to false expression ratios and distort relative expression measurements The ef
105. t cycling conditions 70 Multiplexing 80 Passive reference 77 78 Poor storage 70 Success with PrimeTime assays 45 Melt curves 46 Melting temperature Tm Calculation 30 32 Primers 28 Probes 29 Salt concentration 31 MgCl 44 80 MIQE Guidelines 7 15 82 85 Molecular Beacons 10 Multiplex qPCR 34 Calibration 36 Replicates 36 Validation 34 N Negative controls 38 Normalization 55 Commonly used genes 39 No efficiency correction 55 Reference genes 55 No template control NTC 38 50 5 Nuclease assays 27 38 Design considerations 27 28 See also Assays Design tools aPCR Application Guide Primers 28 Probes 29 Nucleases See also Contamination Contamination 66 DNases 18 RNases 18 O OligoAnalyzer Tool 29 80 84 One step RT qPCR 22 P Passive reference See ROX Positive controls 38 gBlocks Gene Fragments 38 Ultramer Oligonucleotides 38 40 Precision 51 PrimerQuest Design Tool 14 83 Primers Annealing temperature 28 Efficiency 67 Poor efficiency 67 70 GC content 29 Gene specific 21 Integrity 76 Length 29 Melting temperature Tm 28 Oligo dT 22 Random hexamer 21 Random nonamer 21 PrimeTime Predesigned qPCR Assays 14 25 26 See also PrimeTime qPCR Assays Master Mixes 45 Selection tools 82 PrimeTime Predesigned qPCR Assay Selection Tool 27 28 82 83 PrimeTime qPCR Assays 9 14 20 24 25 Avoiding probe degradation 41 Custom assays 25 Custom primer to probe ratios 34 Design 82 Master mixes 44 Multipl
106. t for gene expression efficiency differences one of the following equations should be used This is the recommended method for calculating fold changes in gene expression because it takes into account variation in assay efficiency 28 In equations 4 6 E represents the fold change per cycle per gene For example for an assay with 9596 efficiency E would be 1 4 0 95 or 1 95 E is also called the base of the exponential amplification 56 AXCXIDI INTEGRATED DNA TECHNOLOGIES e Efficiency Corrected based on one sample Eta rget a target control sample Ratio Eren ACgRef control sample equation 4 e Efficiency Corrected based on multiple samples Eta rget Aa target MEAN control MEAN sample Ratio Eref ACgRef MEAN control MEAN sample equation 5 e Efficiency Corrected based on multiple samples and multiple reference genes Etarget 4 lt 4 target MEAN control MEAN sample Ratio Eref mela ACqRef index MEAN control MEAN sample equation 6 Example A researcher used BRCA1 as a target gene and GAPDH as a reference gene After per forming a standard curve with a set of plasmid positive controls the efficiency of each assay was calculated from the slope of the standard curve BRCA Assay Efficiency 93 GAPDH Assay Efficiency 98 The researcher ran the control and treated samples in triplicate which resulted in the following Cg values Cq Mean C
107. t its end value should occur prior to the change in fluorescence and before the amplification curve crosses the threshold Figure 20 Never start the baseline at cycle 1 Default should be 3 but you should always check that the baseline is stable when selecting the start location As depicted in Figure 20 setting the baseline incorrectly can result in either delayed Cg or sloping traces that also have a de layed Cg value See Section 6 2 for more information on setting the baseline 68 XIDT INTEGRATED DNA TECHNOLOGIES Linear Linear Log Rn Baselined ARn Baselined ARn a 5 2 QqD UM amp 2000 START 2 U QqD Me Me O ector A ram gt Piot Rnvs Cyce gt a 5 2 ie WY 2 U eB Me Me O U E Figure 20 Correct and Incorrect Setup of the Baseline Baseline start and stop values in panels A C are set correctly while those in panels D F are set incorrectly with the END point of the baseline setting extending into the exponential part of the curve Incorrect settings result in either delayed Cg values D and F or sloping traces that also have a delayed Cg value 7 2 5 Choice of Dye s Inherent differences in fluorescence intensity of different dyes The height of the ampli fication curve may vary with the reporter dye being used and the optical capability of your instrument The magnitude
108. tions 60 XIDT INTEGRATED DNA TECHNOLOGIES Your curves look like this Instead of this Dye signal Dye signal Rising ROX signal Stable ROX signal You are likely experiencing sample evaporation resulting in rising ROX concentration See Sections 7 8 1 and 7 8 3 Your curves look like this Low height of amplification curve Expected You may be experiencing Dye quenching by proximal G base See Section 4 2 2b Differences in probe concentration See Section 7 2 2 Too much ROX in your sample See Section 7 8 1 a EQ Differences in master mixes used See Section 4 3 2a Signal bleed over See Section 7 4 1 Inherent differences in fluorescence intensity of differ ent dyes See Section 7 2 5 Your curves look like this Observed NTC You are observing true amplificaton in the No Template Control NTC See Section 4 2 6b Threshold 61 aPCR Application Guide Your curves look like this The reaction has poor efficiency See Section 7 3 Several factors can contribute to this Poor primer design Low fluorescent dye intensity and poor instrument Optics Sample inhibition Incorrect primer concentration Your curves look like this You likely have too much probe or insufficient reference dye in your reaction See Section 7 5 You have noisy data 62 63 Your curves look like this Inconsistent spacing Even spacing with 10 fold dilutions Your curves look l
109. tions select 96 or 384 well plates 3 Transfer the appropriate amount of master mix reaction into each well of the reac tion plate 4 Add the cDNA to the appropriate wells Pipette up and down to mix the reaction 5 Sealthe plate with optically clear film 6 Centrifuge the plate at 1000 x g for 30 sec Load the plate on the instrument 7 The suggested real time PCR cycling times are 3 10 min 95 C 40 x 15 sec 95 C 45 sec 60 C This is a typical protocol for qPCR done under standard cycling conditions If a fast master mix is being used itis imperative to adopt the cycling conditions rec ommended by the manufacturer This will result in optimal activation of polymerase present in the master mix The exact cycling parameters will always depend on the particular master mix and instrument being used Refer to the instrument manufac turer s guidelines and the instructions for the master mix you are using for correct cycling conditions Using the correct cycling conditions to activate the enzyme is crucial to the success of the experiment 42 AXCXIDI INTEGRATED DNA TECHNOLOGIES 10X Assay 20 ul reaction 10 ul reaction 5 ul reaction PCR reaction 1 rxn 3 replicates 1 rxn 3 replicates 1 rxn 3 replicates component 10X PrimeTime Assay 2 uL 8 uL 1 uL 4 uL 0 5 uL UE 2X Master Mix 10 uL 40 uL gt HL 20 uL Zo iE 10 uL cDNA RNase free water IDT 8 uL 22 E 4 uL 16 uL 2 uL 8 uL 20X Assay 20 ul reaction 10
110. to the target and not complementary to other sequences Use BLAST to analyze the sequences to ensure their specificity BLAST is a Basic Local Alignment Search Tool provided by NCBI http www ncbi nlm nih gov that finds regions of local similarity between sequenc es Enter an accession number or the sequence in FASTA format and compare it to the genome of a specific species or to all BLAST databases BLAST allows searches against nucleotide or protein databases and provides the statistical significance of the matches for more information see the article Tips for Using BLAST to Locate PCR Primers in the IDT DECODED 1 1 April 2011 newsletter Working with Limited Samples or Low Abundance Targets For two step RI qPCR protocols the input amount of cDNA used for qPCR can be regulated to increase the 27 aPCR Application Guide amount of target available for detection This is useful when working with low abun dance targets when sample is not limited When sample is limited preamplification of RNA by linear isothermal amplification or first strand cDNA prior to qPCR can increase the amount of detectable target for low abundance transcripts from minute amounts of sample This extra step is often incorpo rated when performing single cell analysis or working with clinical samples fine needle biopsies laser captured microdissection samples or FACS generated cells IDT can sup ply custom pooled gene specific primer mixes for this applic
111. ts or patent applications owned by Molecular Probes Life Technologies BHQ quenchers and products incorporating them are to be used for research amp development purposes only and may not be used for any commercial clinical in vitro diagnostic or other use Products incorporating these dyes are subject to the proprietary worldwide rights of Biosearch Technologies Inc and are made and sold under license from Biosearch Technologies Inc There is no implied license for commercial use with respect to the products and a license must be ob tained directly from Biosearch Technologies Inc with respect to any sale lease license or other transfer of the products or any material derived there from the sale lease license or other grant of rights to use the Products or any material derived or produced from them or the use of the Prod ucts to perform services for a fee for third parties including fee for service or contract research CyDye Cy and Cy5 dyes are registered trademarks of GE Healthcare Limited The purchase of this Product includes a limited non exclusive sublicense under U S Patent Nos 5 556 959 and 5 808 044 and foreign equivalent patents and other foreign and U S counterpart applications to use the amidites in the Product to perform research NO OTHER LICENSE IS GRANTED EXPRESSLY IMPLIEDLY OR BY ESTOPPEL Use of the Product for commercial purposes is strictly prohibited with out written permission from Amersham Biosciences Cor
112. ture of oligode oxyribonucleotides on the fluorescent properties of conjugated dyes Nucleic Acids Res 30 9 2089 2195 Tyagi S and Kramer FR 1996 Molecular beacons probes that fluoresce upon hybridization Nat Biotechnol 14 3 303 308 VanGuilder HD Vrana KE and Freeman WM 2008 Twenty five years of quantitative PCR for gene expression analysis Biotechniques 44 5 619 626 Marras SA Kramer FR and Tyagi S 1999 Multiplex detection of single nucleotide variations using molecular beacons Genet Anal 14 5 6 151 156 Gudnason H Dufva M et al 2007 Comparison of multiple DNA dyes for real time PCR ef fects of dye concentration and sequence composition on DNA amplification and melting temperature Nucleic Acids Res 35 19 e127 Sang F and Ren J 2006 Capillary electrophoresis of double stranded DNA fragments using a new fluorescence intercalating dye EvaGreen J Sep Sci 29 9 1275 1280 Zipper H Brunner H et al 2004 Investigations on DNA intercalation and surface binding by SYBR Green its structure determination and methodological implications Nucleic Acids Res 32 12 e103 Boom R Sol CJ et al 1990 Rapid and simple method for purification of nucleic acids J Clin Microbiol 28 3 495 503 Fleige S and Pfaffl MW 2006 RNA integrity and the effect on the real time qRT PCR perfor mance Mol Aspects Med 27 2 3 126 139 Nolan T Hands RE and Bustin SA 2006 Quantification of mRNA usin
113. uding knowledge of the transcript variants and their exon organization Use databases such as Ensembl or GenBank to identify exon junctions splice variants and locations of single nucleotide polymorphisms SNPs For genes that have multiple tran script variants align related transcripts to understand exon overlap using a program such as Clustal or use NCBI online tools such as the Gene Viewer For transcript specific designs target primers and probes within exons unique to the transcripts of interest and BLAST primer and probe sequences to ensure they do not occur in related transcripts or cross react with other genes within the species see below For splice common designs target primers and probes within exons found across all transcript variants Note that the PrimeTime Predesigned qPCR Assay Design Tool takes these factors into consideration for you see Section 8 2 1a Avoid SNPs With the increased focus on high throughput sequencing the number of identified SNPs in the human genome is rapidly increasing In the human genome SNPs are present in at least 196 of the population and occur on average once every few hun dred bases A single mismatch between primer and target due to a SNP can significantly decrease the melting temperature of that hybrid by up to 10 C affecting the efficiency of the PCR and ultimately the interpretation of experimental results Ensure Specificity Ensure that both the primers and the probe are specific
114. ul reaction 5 ul reaction PCR reaction 1 rxn 3 replicates 1 rxn 3 replicates rxn 3 replicates component 20X PrimeTime Assay 1 uL 4 uL 0 5 uL 2 0 pL 0 25 uL 1 uL 2X Master Mix 10 HE 40 uL gt HL 20 uL 2 5 pit 10 uL cDNA RNase free water IDT O Ul 36 uL 4 5 uL 18 uL 2 29 PL 9 uL 40X Assay 20 ul reaction 10 ul reaction 5 ul reaction PCR reaction 1 rxn 3 replicates 1 rxn 3 replicates 1 rxn 3 replicates component 40X PrimeTime Assay 0 5 UL 2 uL 0 25 uL 1 uL 0 13 uL 0 5 HL 2X Master Mix 10 uL 40 uL 5 uL 20 uL 23 uL 10 uL cDNA RNase free water IDT 9 ok 38 uL 4 75 uL T9 uL 2 So UL 2 2 We Replicates include excess to account for volume loss during pipetting IDT recommends 1 100 ng of cDNA per 20 uL reaction t DEPC free nuclease free IDT water is recommended It is imperative that every single sample be treated in the same manner in order to achieve reliable results For that reason it is very important to use a mix to ensure that every sample receives the same amount of each ofthe reaction components See Figure 15 for information on all master mixes tested at IDT Table 6 Master Mix Volumes for Number of Assays Planned 43 aPCR Application Guide 4 3 2a Master Mixes Master mixes prepared by manual addition of components reaction buffer dNTPs MgCl and Taq polymerase allow maximum flexibility because components can be adjusted according to experimental needs However most researchers use commerci
115. uplex A PrimeTime HPRT and GUSB qPCR Assays Assay IDs Hs PT 58v 45621572 and Hs PT 58v 27737538 respectively 40 HPRT FAM labeled probe Singleplex hw 30 9 3 E gt 0 P 9 15 10 0 B3 0 001 5 10 1 20 2 Cycle 40 GUSB VIC labeled probe 35 Singleplex 30 xx j 25 c 8 20 po o 0 5 10 15 20 Cycle B Inventoried HPRT and GUSB Assays from Supplier A Duplex FAM dye Efficiency 95 81 R 0 9993 Singleplex Efficiency 91 10 R 0 9969 Log Singleplex D Duplex Duplex VIC dye Efficiency 96 11 R 0 9995 Singleplex Efficiency 96 68 R 0 9986 Figure 15 Successful Singleplex and Duplex Amplification Using PrimeTime Gene Expression Master Mix All PCRs contained the PrimeTime Gene Expression Master Mix with reference dye and were run on a 7900HT Real Time PCR System Thermo Fisher Scientific with PCR efficiencies between 90 1 1096 A Singleplex and duplex PCRs included PrimeTime qPCR HPRT and GUSB Assays and gBlocks Gene Fragments template 107 10 copies B Singleplex and duplex PCRs included inventoried HPRT and GUSB qPCR assays Supplier A and cDNA template 50 0 0032 ng 45 aPCR Application Guide 5 Assay Validation Before valuable experimental samples are consumed in the qPCR analysis phase it is im portant to have carefully evaluated the assay performance for specificity and efficiency It is important to validate each new assay eve

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