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1. Methods Quantitative real time PCR protocol for analysis of nuclear receptor signaling pathways Angie L Bookout and David J Mangelsdorf Nuclear Receptor Signaling The Open Access Journal of the Nuclear Receptor Signaling Atlas Corresponding Author davo mango utsouthwestern edu Howard Hughes Medical Institute Department of Pharmacology University of Texas Southwestern Medical Center Dallas TX USA A major goal of the Nuclear Receptor Signaling Atlas NURSA is to elucidate the biochemical and physiological roles of nuclear receptors in vivo Characterizing the tissue expression pattern of individual receptors and their target genes in whole animals under various pharmacological conditions and genotypes is an essential component of this aim Here we describe a high throughput quantitative real time reverse transcription PCR QPCR method for the measurement of both the relative level of expression of a particular transcript in a given tissue or cell type and the relative change in expression of a particular transcript after pharmacologic or genotypic manipulation This method is provided as a standardized protocol for those in the nuclear receptor field It is meant to be a simplified easy to use protocol for the rapid high throughput measurement of transcript levels in a large number of samples A subsequent report will provide validated primer and probe sequence information for the entire mouse and human nuclear receptor su
2. using mouse PNR are shown in Figure 3 The slope of the standard curve is indicative of PCR efficiency formula 1 Figure 4 given by formula 2 Figure 4 Applied Biosystems 2001 The slope can be affected by template quality pipetting errors etc and for this reason a Standard curve for the endogenous reference gene is always run on the same plate The difference in slopes of the reference gene and the gene of interest should be less than 0 1 Applied Biosystems 1997 Once a primer set has been validated using the SYBR Green chemistry switching to the TaqMan chemistry is as simple as adding the probe at 250nM final concentration and switching to the buffer optimized for use with TaqMan probes ABI cat 4324018 QPCR Assay Preparation The QPCR assay is run in a 384 well Optical Reaction Plate with 10ul final volume per well Each sample is run in triplicate for each gene to be assayed The reaction consists of 25ng template cDNA 1X SYBR Green PCR Q PCR analysis of nuclear receptor expression MasterMix and 150nM each of forward and reverse primer Reaction components are pre mixed in 8 well PCR tube strips and then pipetted with a multi channel pipette into the reaction plate Premixing the components decreases the amount of user introduced variation by ensuring a homogeneous mix of template and primers and also by minimizing the number of pipettes which can introduce bubbles and or aerosol contaminants into the
3. capacity its potential for use with robotics and its relatively widespread availability in the field The 7900HT is a rapid cycling instrument with a single run lasting approximately 2 hours The instrument allows the use of either 96 well or 384 well plates The protocols employed herein use the 384 well format The instrument also allows for the addition of a robotic plate loader arm which enables unattended operation throughout the day RNA and cDNA preparation RNA STAT 60 was obtained from Tel Test Inc Friendswood TX cat CS 502 RNase free DNase was obtained from Roche Molecular Biochemicals Indianapolis IN cat 776785 1X First Strand Buffer 10mM DTT and SuperScript RTII reverse transcriptase were obtained as a kit from Invitrogen Carlsbad CA cat 18064 014 2mM dNTPs were obtained from ABI cat N808 007 and 0 08ug ul random hexamers from Roche Molecular Biochemicals cat 1034731 QPCR Assay 384 well Optical Reaction Plate are obtained from ABI cat 4309849 Sybr Green PCR MasterMix is obtained from Sigma St Louis MO cat 84438 8 well PCR tube strips are obtained from CLP San Diego CA cat 3426 8A and the optical adhesive cover from ABI cat 4313663 or 4311971 Methods SYBR Green I versus TaqMan based Assays There are two types of fluorescence monitoring chemistries available for use on the ABI 7900HT SYBR Green and TaqMan Applied Biosystems 2003 The first employs
4. mice fed control vehicle or one of a number of typical nuclear receptor ligands Q PCR analysis of nuclear receptor expression pe relative fold change oO 60 Kidney SREBP1c mean average J B a RXR LXR RXR PPARa PPARy FXR LXR relative fold change Figure 6 Typical experiment in which wild type male mice were treated with several different nuclear receptor agonists The relative changes in mRNA expression for known receptor target genes in this case SREBP 1c an LXR target gene were measured using the comparative Ct method a SREBP 1c expression plotted for each of the animals S D Each color represents a different experimental group n 4 animals 0 SREBP 1c expression plotted as averages of the fold changes for the 4 animals in each treatment group shown in a SEM Note that the SREBP 1c transcript increases relative to control VEH in the animals treated with an RXR agonist LG268 an LXR agonist T1317 or both LG269 and T1317 together T LG Note that ligands for PPAR a fenofibrate PPAR y troglitazone FXR CDCA PXR PCN and CAR TCPOBOB have no effect on the level of the SREBP 1c message relative to control Discussion Here we have described a simple to use yet effective real ttme QPCR methodology The protocols and the worksheets provided are intended to introduce and standardize the assay for researchers in the nuclear receptor field but may be applied to any field of study I
5. northern blots However because of its very high expression the concentration of starting template in a QPCR assay must be low and the detection of the gene of interest may be lost at the lower template quantity The choice of the endogenous reference therefore should be determined empirically and based on the system under study Primers Perhaps the most important factor of QPCR analysis is the choice and validation of primers The oligonucleotide sequences must be highly specific which may be difficult when dealing with isoforms or splice variants A primer set that has been used in conventional end point analysis PCR may not be sufficient for quantitative measurements The primer set must pass a set of validation parameters as described later One drawback of QPCR over northern analysis is that QPCR cannot detect the quality i e the size of an RNA message the number of differently sized messages e g splice variants or their expression levels relative to each other One way around this problem is to design sub type specific primer sets but unfortunately this strategy still does not result in any information concerning the overall quality of the transcripts For this reason it is worth investing the time to confirm the quality of the template RNA by northern analysis against a known transcript Quantitative Assay Types Each of the following sections describes the detailed methodology for performing both SYBR Green an
6. priming wopuel random Wwopue random WOpue 1 random AAAAAAA random 6 bases random 6 bases random 6 bases polyA tail Figure 2 Representation of RNA priming for reverse transcription See text for more details RNA and cDNA Preparation Please refer to the sections Q PCR supplies reagents and stock solutions and Preparation of cDNA Standards in the Supplementary File 1 for the detailed protocols and worksheets for this section Total RNA is prepared from cells or frozen tissues with RNA STAT 60 according to the manufacturer s instructions For a typical assay 4ug of total RNA is treated with a 1 5 dilution of RNase free DNase in the presence of 4 2mM MgCl in a final volume of 20ul This is performed in 0 2ml thin walled PCR tubes in a standard thermocycler at 37 C 30 min 75 C 10 min and 4 C hold The reverse transcription mix consisting of 1X First Strand Buffer 10mM DTT 200U of SuperScript RTII reverse transcriptase 2mM dNTPs and 0 08ug ul random hexamers is then added directly to the tubes with the DNAse treated RNA for a final volume of 100ul The cDNA synthesis is carried out in the thermocycler at 25 C for 10 min 42 C for 50 min 72 C for 10 min and 4 C hold Following the reverse transcription DEPC treated H20 is added to the unknown samples to bring the volume to 200ul and the cDNA concentration to 20ng ul Note that samples used as cDNA standards are not diluted prior to m
7. the DNA intercalating dye SYBR Green as the reporter fluorophore It works like ethidium bromide by binding double stranded DNA which is the product of the PCR As the reaction cycle progresses the instrument monitors and records the increase in fluorescence over time The SYBR Green assay only requires a validated primer pair in addition to the regular PCR components The TaqMan chemistry utilizes FRET fluorescence resonance energy transfer technology It requires the use of an additional type of primer called a probe which is labeled with a fluorescent reporter dye on its 5 end and a 3 quencher dye While the probe is still intact the 5 and the 3 dyes are in close proximity and the fluorescent signal is quenched via FRET As the PCR product is synthesized the probe which sits on a specific sequence in a region between the forward and reverse primers is cleaved by the nuclease activity of the Taq polymerase As the 5 reporter dye is released it continuously fluoresces One probe is cleaved for every www nursa org NRS 2003 Vol 1 DOI 10 1621 nrs 01012 Page 1 of 7 Methods PCR product made during the reaction and the machine records the concomitant fluorescence increase over time There are pros and cons to each of the chemistries employed for QPCR One advantage of SYBR Green over TaqMan is that the initial assay preparation only requires a few days for primer design and validation T
8. age is determined given by formula 4 Figure 4 A single outlier point that has a www nursa Oorg NRS 2003 Vol 1 DOI 10 1621 nrs 01012 Page 5 of 7 Methods value gt 17 CV may be removed from the calculations To determine the mRNA level in each unknown sample the gene of interest is normalized to the reference gene e g 18S rRNA to account for cDNA loading differences and calculated using formula 5 Figure 4 The resulting normalized values are plotted as a bar graph the standard deviation formula 6 Figure 4 given by formula 7 Figure 4 An example of LXR a expression in 38 mouse tissues is shown in Figure 5 signal 18S rRNA S Se e eee ae Ky S gt PO 4 y A Vo Prg 2 GS Ss Ry S s K g Re SX lt g gt gt A y Po amp PP LA ESE SFS KY ASF OWL DPF FSA VCS FS Q ee N ESS os sy e Ss LS SPO 5 SS S se Ca S 2 gt Wo Figure 5 Expression profile for mouse LXR generated using the standard curve method The relative levels of the mRNA transcript are shown S D from triplicate readings 18S rRNA was used as the normalizer gene so that the level of LXR a may be compared between tissue types Comparative Ct or AACt Method Please refer to the sections SYBR Green AACt QPCR Assay Worksheet and TaqMan AACt QPCR Assay Worksheet in Supplementary File 1 for the detailed protocols and worksheets for this section For each of the 3 replicates of a sample the average cycle
9. aking the 5 fold dilution series used in primer validation and standard curve assays This will result in enough template cDNA to test approximately 40 QPCR targets www nursa org NRS 2003 Vol 1 DOI 10 1621 nrs 01012 Page 3 of 7 Methods Amplification Plot Dissociation Curve Dipsusigtisns Ca Q PCR analysis of nuclear receptor expression Standard Curve Plot Standard Plot 1061 Quantity Standarda Pin o M Standard Curve Plot Detector ftest Standard Plot Amplification Plot a 100062 Amplification Plot AAA AE ee E fy VY i ARKAA g gt lt f g f Fr J XI E A f N bi j i Cycle Detect Z Plotfazn vs Threshold 0 171355 Amplification Plot jan ta Amplification Plat OS A 7 P RE a s IAA P p 4 F Q A A A lt j o ty Mb I VA BLY a Mat IF AY V Y Y 2 Cycle Detector htest 8 Z Plotfarnvs Cycle Threshold 01249609 Amplification Plot C Amplification Plot 1 000 Er oc a a 1000 5 5 2 a 1 J Cycle fi Detectorfiest2 m PlotfaRnvs cycle Thnreshold 0 07963643 i F Temasa nese o ST OO noS DOAN Quantity Figure 3 Primer set validation a Example of a valid primer set for mouse LXR a using SYBR Green The presence of a single peak in the dissociation curve and the 3 3 slope and 0 99 R2 value of the standard curve plot
10. aqMan requires the additional synthesis of the dual labeled probe after the validation of the potential primer set using SYBR Green The probe synthesis usually takes 2 3 weeks and is expensive relative to the cost of the primers The increasing availability of validated TaqMan primer probe sets from several vendors may eventually bring the costs of this method down In general TaqMan has been considered to be more sensitive when detecting low copy numbers lt 10 copies because of its ability to resolve the signal of a single copy of template Wittwer et al 1997 However in most cases the sensitivity of the instrument is limited to gt 10 copies of template due to stochastic effects Morrison et al 1998 SYBR Green may have a slight edge in sensitivity at gt 10 copies because the reporter dye binds to any double stranded DNA present in the reaction and does not require a probe cleavage event for the fluorescence detection as does TaqMan The result is detection of the PCR product at earlier cycles Wittwer et al 1997 This is especially important in the case of low abundance transcripts gt 10 copies where the number of PCR cycles required for fluorescence detection above background might be beyond the range of cycles in the TaqMan but not the SYBR assay The double stranded DNA binding property is also a disadvantage of SYBR Green over TaqMan because non specific products and mRNAs with high sequence identit
11. are indicative of a good set of primers b Example of an invalid primer set for mouse HSD due to the amplification of non specific products as indicated by the presence of multiple peaks in the dissociation curve This may be an effect of non specific priming or primer dimerization Note that the slope of the standard curve plot 3 2 is within the acceptable range of a valid primer set but the dissociation curve renders this set of primers unacceptable c Example of an invalid primer set for mouse PNR due to poor amplification Note the unacceptable slope 2 28 in the standard curve plot and the presence of multiple peaks in the dissociation curve QPCR Primer Design for Assays using the ABI 7900HT QPCR assays rely on a set of universal cycling conditions The thermocycle as well as the buffer conditions MgClo salts dNTPs Tag DNA polymerase will always be the same One of the few parameters that does vary is the QPCR primer set Because of this the design and the pre validation of the primers are essential to generate reliable data The nucleotide sequence and mRNA exon structure for each gene of interest are obtained from the NCBI Locus Link database NCBI 2003 Ideally the reference sequence RefSeq is obtained and used for primer design If a RefSeq is not available the GeneBank entry that gives an NM_ or complete cds is used If the mRNA to genomic alignment is not available from Locus Link a BLAST search of t
12. d TaqMan assays Figure 1 depicts the order of events necessary to employ these techniques Isolate total RNA t Validate Design amp validate SYBR Green primer LET LUETLKA o go ef Perform TaqMan assays Figure 1 Typical work flow for designing and implementing Real Time PCR assays See text for more details Perform SYBR i Green assays There are three quantitative methods the absolute standard curve the relative standard curve and the comparative cycle time Ct methods Applied Biosystems 2003 Herein we describe methods for the relative standard curve and the comparative Ct also called ACt www nursa org NRS 2003 Vol 1 DOI 10 1621 nrs 01012 Page 2 of 7 Methods methods For discussion concerning the absolute standard curve method Applied Biosystems 1997 The relative standard curve method is used to assess the amount of a particular transcript in a group of samples lt is comparable to a multiple tissue northern blot It requires that a template dilution series of a cDNA standard be included for each gene on each plate For relative quantitation the template concentration values are arbitrary Quantities interpolated from the resulting standard curve are used to calculate relative mRNA levels in each unknown sample The comparative Ct method or ACt is used to assess relative changes in MRNA levels between two samples and does not require the use of the tem
13. he mRNA against the proper genome can be used to determine intron exon boundaries For both SYBR Green and TaqMan based assays Primer Express Software Applied Biosystems is used to design the TagMan MGB Probe and Primer sets The software returns a list of primer and probe sequences as matched primer probe sets and primers are chosen based on their binding sites In order to amplify only mRNA and not genomic DNA the PCR product or amplicon should span the intron junction between two exons Ambion 2001 When using TaqMan ideally the probe should sit across the junction Minimally each primer should sit in completely separate exons In some cases the software may not return any sets that meet these criteria initially In this case two design parameters in the software may be adjusted amplicon length and amplicon melting temperature Tm The amplicon length should be a minimum of 50 base pairs and maximum of 150 base pairs and if necessary should be adjusted in 10 base pair increments Amplicon T s usually start at 85C and can be adjusted upward to a maximum of 95C especially in the case of GC rich target sequences Once the primers have been chosen a general BLAST of each primer sequence Is run to ensure their unique specificity Results Initial Validation of Primer Sets Please refer to the sections SYBR Green Template Titration Assay and TaqMan Template Titration Assay in Supplementa
14. le brain embryo heart intestine kidney liver lung muscle ovary pancreas placenta macrophage skin testis and white adipose tissue The assay consists of a 5 fold dilution series of CDNA reverse transcribed from the universal RNA 50ng 10ng 2ng 0 4ng 0 08ng 0 016ng and 2 control samples a no template control NTC and a no reverse transcriptase RT control Amplification of the NTC sample indicates the presence of primer dimer formed during the reaction The difference in cycle time between the NTC and the experimental samples must be greater than seven to ensure that the assay is monitoring specific fluorescence of the experimental sample personal communication ABI The RT sample is included to confirm the absence of genomic amplification After the template titration assay run is complete the ABI 7900HT instrument software SDS2 1 will plot a standard curve and a dissociation curve for each target gene A valid primer set should have a slope of 3 3 anda correlation coefficient R2 value gt 0 95 for the standard curve In addition the dissociation curve should appear as a single stacked peak at the amplicon Tm determined by the Primer ExpressTM software Examples of a typical standard curve plot for a valid primer set e g using mouse LXR an invalid primer set due to non specific priming e g using mouse hydroxysteroid dehydrogenase HSD and an invalid primer set due to poor amplification e g
15. n a subsequent publication a complete set of QPCR primers and TaqMan probes for all mouse and human nuclear receptors will be reported Supplementary Material Supplementary File 1 Q PCR Protocols and Worksheets Acknowledgements We thank all current and former lab members for invaluable critique and discussions concerning the development of these techniques in addition to the labor involved with the animal studies We also thank Applied Biosystems for the technical support Any questions or suggestions concerning the downloadable materials are encouraged and should be addressed to Angie Bookout at abookout biochem swmed edu DJM is an investigator of the Howard Hughes Medical Institute This work was funded in part by the NIH NURSA grant number NIDDK U19DK62434 References Ambion 2001 The Top 10 Most common quantitative PCR pitfalls Technotes Newsletter 8 http www ambion com techlib basics rtpcr topten html Applied Biosystems 1997 Relative Quantitation Of Gene Expression ABI PRISM 7700 Sequence Detection System User Bulletin 2 Rev B Applied Biosystems Applied Biosystems 2001 ABI Prism 7900HT User Manual www nursa org NRS 2003 Vol 1 DOI 10 1621 nrs 01012 Page 6 of 7 Methods Applied Biosystems 2003 Essentials of Real Time PCR http docs appliedbiosystems com pebiodocs 00105622 pdf Morrison T B Weis J J and Wittwer C T 1998 Quantification of low copy transcripts by con
16. optical plate itself Applied Biosystems 2001 The plate is then covered with an optical adhesive cover and centrifuged to bring the liquid to the bottom of the wells of the plate 4 Ci ave GOI qiy avg 5 normalized value ee reference qty avg 6 S D 7 S D normalized value x ACV of reference CV of GOI B AC 9 ACI avg Clay avg Ci 10 stdev n stdev of reference stdev of GOTY 11 AAC ACI ay ACi ai 12 stdev 4 T n 2 stdev Wan as stdev of group 13 SEM dev OF Prey of samples in group Figure 4 Formulas for Q PCR calculations See text for more details Data Analysis Standard Curve Method Please refer to the sections SYBR Green Standard Curve QPCR Assay Worksheet and TaqMan Standard Curve QPCR Assay Worksheet in Supplementary File 1 for the detailed protocols and worksheets for this section The instrument software calculates the quantity of transcript in each unknown sample based on the linear regression formula of the standard curve and data are exported as a tab delimited text file Further data analyses are done using Microsoft Excel or another comparable program For each sample the quantity of the gene of interest GOI and the reference gene reference are determined in triplicate and from these values the average transcript quantity avg the standard deviation of the average stdev and the coefficient of variation formula 3 Figure 4 of the aver
17. perfamily Received Novemeber 4th 2003 Accepted December 12th 2003 Published December 23rd 2003 Abbreviations GAPDH Glyceraldehyde 3 phosphate dehydrogenase GOI Gene of interest GSRP Gene specific reverse primer PCR polymerase chain reaction QPCR Quantitative real time PCR Tm Amplicon melting temperature Copyright 2003 Bookout and Mangelsdorf This is an open access article distributed under the terms of the Creative Commons Non Commercial Attribution License which permits unrestricted non commercial use distribution and reproduction in any medium provided the original work is properly cited Cite this article Nuclear Receptor Signaling 2003 1 e012 Introduction Characterizing the tissue expression pattern of individual receptors and their target genes in whole animals under various pharmacological conditions and genotypes is one of the aims of the Nuclear Receptor Signaling Atlas NURSA Here we describe a high throughput quantitative real time reverse transcription PCR QPCR method for the measurement of both the relative level of expression of a particular transcript in a given tissue or cell type and the relative change in expression of a particular transcript after pharmacologic or genotypic manipulation Reagents and Instruments Thermal cycler Although several instruments are now available we chose to use the Applied Biosystems ABI Foster City CA 7900HT instrument because of its high throughput
18. plate dilution series It does however require that the amplification efficiencies of the genes to be compared described later be the same Applied Biosystems 1997 Cycle times rather than interpolated template quantities are used for the calculations An example of employing the comparative Ct method would be the comparison of the expression of a nuclear receptor target gene in wild type vs receptor null animals or ligand treated vs untreated animals One step vs Two step Reverse Transcriptase PCR Reverse transcription RT is the enzymatic process by which complementary DNA cDNA is synthesized by an RNA dependent DNA polymerase i e reverse transcriptase It requires a small oligonucleotide DNA primer There are several types of primers including gene specific reverse primers GSRP oligo d Ts and random hexamers also called p d 6 The GSRP is specific to the cDNA to be synthesized and may be used to selectively RT a specific RNA transcript Oligo d Ts consist of short stretches of thymines and selectively target RNAs with a polyA tail or any polyA tract Random hexamers are mixtures of 6 base pair primers with varying sequences They randomly bind their complementary RNA sequences A schematic representation of the various types of priming for reverse transcription is shown in Figure 2 cDNA is synthesized in one or two steps One step RT PCR is done in a single reaction in which the reaction mixture contains
19. ry File 1 for the detailed protocols and worksheets for this section Oligonucleotides are purchased from a commercial vendor at the small scale synthesis with the minimum purification We purchase our primers from Integrated DNA Technologies at 25nM scale with standard desalting We have found that additional purification such as HPLC is not necessary and only adds to the cost of each primer The small scale synthesis also adds to the savings especially in the case that the primers do not work initially The oligonucleotides are resuspended to 100uM in sterile filtered ddH5O and an aliquot is diluted to 2 5uM The forward and reverse 2 5uM primers are then mixed at a 1 1 ratio for use in subsequent assays In the past www nursa org NRS 2003 Vol 1 DOI 10 1621 nrs 01012 Page 4 of 7 Methods matrix tests in which several combinations of different forward and reverse primer concentrations are assayed were used to determine optimal final primer concentrations Morrison et al 1998 We have found that this is no longer necessary as 150nM final concentration each of forward and reverse primers has proven to be a universal condition in our assays To validate the primers a template titration assay is done For human transcripts we use the Universal Reference Total RNA from BD Clontech Palo Alto CA For mouse transcripts a mixture of equal quantities of RNA from each of the following tissues is used adrenal who
20. the reverse transcriptase and the PCR polymerase Here both the RT primer and one of the PCR primers is a GSRP Two step RT PCR involves two separate reactions for the RT and the PCR and the RT primer is either oligo d Ts or random hexamers The RT reaction is carried out at 42 C to reduce or inhibit the occurrence of RNA secondary structure For quantitative RT PCR QPCR random hexamers are used to ensure the efficient amplification of all RNAs in a sample Because the primer sequences are random and therefore can bind anywhere along the RNA there is avery high probability that all transcripts will be reverse transcribed at near 100 RT efficiency ABI personal communication When using oligo d Ts only RNAs Q PCR analysis of nuclear receptor expression containing a polyA sequence will be amplified and incomplete or partially degraded RNA will not be detected in the subsequent QPCR assay Use of the GSRP appears attractive at first since a specific target will be amplified This can be potentially problematic however because the RT efficiency of each transcript has to be taken into consideration Also RNA secondary structure may prevent the efficient RT of a specific target and would also have to be taken into consideration Gene specific priming dySsoS GSRP specific gene seq AAAAAAA polyA tail Oligo dT priming EERE AAAAAAA internal polyA tract JURE AAAAAAA polyA tail Random hexamer
21. time Ct the standard deviation stdev and the coefficient of variation formula 3 Figure 4 is calculated given by formula 4 Figure 4 Outlier points gt 4 CV are removed from the calculations For each sample the average Ct of the GOI avg Ctgo is normalized to the average Ct of the reference gene avg Ct e for the same sample to calculate the normalized Ct for the GOI formula 8 Figure 4 using formula 9 Figure 4 The standard deviation of the ACt is calculated using formula 10 Figure 4 The calibrator is chosen to be the sample the tissue the gene or the control group to which the others will be compared For example in an experiment in which wild type mice are compared to knockout mice the calibrator would be the wild type mouse sample The AACT or calibrated value for each sample is given by formula 11 Figure 4 The stdev jac is the same as stdev act The fold induction for each sample relative to the calibrator 2 The resulting induction values are usually plotted as a bar graph The measure of experimental error is the standard deviation of the mean stdeVfoid change given by formula 12 Figure 4 If there are multiple samples in multiple treatment groups the average the fold induction for each group is plotted The biological variance is the standard error of the mean given by formula 13 Figure 4 An example is shown in Figure 6 where the induction of SREBP 1c expression is compared between wild type
22. tinuous SYBR Green monitoring during amplification Biotechniques 24 954 8 NCBI 2003 Locus Link http www ncbi nim nih gov LocusLink Suzuki T Higgins P J and Crawford D R 2000 Control selection for RNA quantitation Biotechniques 29 332 7 Wittwer C T Herrmann M G Moss A A and Rasmussen R P 1997 Continuous fluorescence monitoring of rapid cycle DNA amplification Biotechniques 22 130 1 Q PCR analysis of nuclear receptor expression www nursa org NRS 2003 Vol 1 DOI 10 1621 nrs 01012 Page 7 of 7
23. y may be detected One additional advantage of TaqMan is that the probe offers an added layer of specificity in addition to the forward and reverse primers The probe sequence must exactly match the target sequence to which it binds as a single nucleotide difference in the probe sequence will prevent the cleavage event necessary to generate a reporter signal Endogenous Normalizer One of the experimental controls included in a gene expression assay is the loading or internal control It is used to normalize the signal value of each sample so that the differences between samples are the result of a real biological difference and not because of inconsistent loading Housekeeping genes are the typical choice due to their mostly consistent expression levels in all cell types Glyceraldehyde 3 phosphate dehydrogenase GAPDH B actin cyclophilin and 18S rRNA are commonly used Both GAPDH and B actin have been shown to vary with numerous conditions Suzuki et al 2000 and are not the best choice Cyclophilin expression is equal among most tissues with the exception of heart and muscle unpublished observation These tissues show lower levels of the transcript relative to other tissues so the normalized values for the transcript under study may be exaggerated 18S may be the preferred standard because of its relatively invariant Q PCR analysis of nuclear receptor expression level among tissues and treatment conditions also observed in

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