A practical approach to assay design for qPCR

A Practical Approach to Assay Design for qPCR Overcoming Difficult Assays, Designs andOptimization while Conforming to the MIQE Guidelines Francisco Bizouarn International Field Application Specialist Gene Expression Division Bio-Rad Laboratories

A new beginning.AMPLIFICATION www.bio-rad.com/genomics/pcrsupport

What is MIQE? It’s a ChecklistAMPLIFICATION • qPCR community driven guidelines for essential and desired information in litterature; – Experimental Design – Sample Information – Nucleic Acid Extraction – Reverse Transcription – qPCR Target Information – qPCR Oligonucleotides – qPCR Protocol – qPCR Validation – Data Analysis www.bio-rad.com/genomics/pcrsupport

Generating a good assay is easyAMPLIFICATION • Following a few simple steps: – Design assay – Run a gradient – Run a dilution series to validate assay dynamic range • Meeting MIQE guidelines requires very little additional effort. – Target Information – Oligonucleotide information – Protocol – qPCR Protocolalidation www.bio-rad.com/genomics/pcrsupport

What is MIQE? It’s a ChecklistAMPLIFICATION • qPCR community driven guidelines for essential and desired information in litterature; – Experimental Design – Sample Information – Nucleic Acid Extraction – Reverse Transcription – qPCR Target Information – qPCR Oligonucleotides – qPCR Protocol – qPCR Validation – Data Analysis www.bio-rad.com/genomics/pcrsupport

Assay designAMPLIFICATION • Often oversimplified by the use of software or by many companies that offer design services. • Design a critical parameter. • Following a few simple steps will increase the chances of designing a successful assay. • Let’s use an example: target CCL26 in HUVEC cells www.bio-rad.com/genomics/pcrsupport

CCL26 cDNA sequenceAMPLIFICATION CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT TGACTTGTTC AT www.bio-rad.com/genomics/pcrsupport

Sequence Alignment (BLAST)AMPLIFICATION • Prior to designing primers, it’s a good idea to run a sequence homology analysis. (BLAST) • This allows the identification of sequences that may co- amplify or interfere with our intended target. • The data is freely available, so why not make use of it. • http://blast.ncbi.nlm.nih.gov www.bio-rad.com/genomics/pcrsupport

CCL26 with homologous sequencesAMPLIFICATION CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT TGACTTGTTC AT www.bio-rad.com/genomics/pcrsupport

2nd structure analysis of CCL26AMPLIFICATION • DNA is often seen as a linear polymer. • In it’s single stranded state (cDNA) regions that have complimentary sequences will tend to hybridize generating hairpins that may inhibit primer annealing. • Avoiding these sequences when possible will improve amplification effiecency. • http://mfold.bioinfo.rpi.edu/cgi-bin/dna- form1.cgi www.bio-rad.com/genomics/pcrsupport

CCL26 with 2nd structuresAMPLIFICATION CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT TGACTTGTTC AT www.bio-rad.com/genomics/pcrsupport

Amplicon sizeAMPLIFICATION • Classic qPCR rules dictate that amplification products be between 75 and 200 bp in length. • These limits are not absolute. It is better to design a larger amplicon than to risk target specificity and primer annealing issues • New “ultra fast” reagents allow much larger amplicons to be used in qPCR. www.bio-rad.com/genomics/pcrsupport

Design primersAMPLIFICATION • Some primer design packages will take both sequence homology and secondary structure issues into account when designing assays. • Due to the restrictions imposed on the design software, they can fail. • Although not recommended, designing assays by “thumb” can be performed. GCGGAATCTT TTCTGAAGGC TACATGGACC • There are also databases of freely available primers and probes that have been previously tested. www.bio-rad.com/genomics/pcrsupport

qPCR Target InformationAMPLIFICATION www.bio-rad.com/genomics/pcrsupport

qPCR OligonucleotidesAMPLIFICATION www.bio-rad.com/genomics/pcrsupport

CCL26 primer designAMPLIFICATION CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT TGACTTGTTC AT www.bio-rad.com/genomics/pcrsupport

Using Thermal GradientsAMPLIFICATION • Thermal optimization is often the first parameter an individual using PCR will test to get the optimal reaction conditions. • Unfortunately many qPCR users often ignore this parameter, as though antiquated, in favor of more elaborate primer design software packages. • Finding the correct annealing temperature at which to run an assay is critical. www.bio-rad.com/genomics/pcrsupport

Using Thermal GradientsAMPLIFICATION • 40 wells @ 5 ul each • Prepare a master-mix for 40 wells • 100 ul 2X Supermix • Primer concentration typically between 200 and 500nM • ul forward primer (300nM) • ul reverse primer (300nM) • ul DNA or cDNA • Critical parameter: amount of DNA or cDNA used. Use as little • ul H20 as possible. --------- • 200 ul total Vortex! www.bio-rad.com/genomics/pcrsupport

Assay optimizationAMPLIFICATION For 1 Rev 1 5’ 3’ For 2 Rev 2 For 1 For 2 Rev 1 Rev 2 10o above design { 5o below design www.bio-rad.com/genomics/pcrsupport

Gradient analysisAMPLIFICATION CCl26 amplified using Bio-Rad iQTM SYBR® Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport

Gradient analysisAMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport

Optimal Annealing RangeAMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport

Effect of Annealing Temp on C(t)AMPLIFICATION C(t) vs Annealing Temp 72 70 68 66 Annealing Temp 64 62 60 58 56 54 52 25 30 35 40 45 50 55 C(q) CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport

Different reagents behave very differentlyAMPLIFICATION C(t) vs Annealing Temp C(t) vs Annealing Temp 72 72 70 70 68 68 66 66 Annealing Temp Annealing Temp 64 64 62 62 60 60 58 58 56 56 54 54 52 52 25 30 35 40 45 50 55 25 30 35 40 45 50 55 C(q) C(q) CCl26 amplified using Bio-Rad Sso Fast EVA Green Supermix: CCl26 amplified using Other Reagent A: 5ul Assay 5ul Assay98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt 95oC 5min / 50x 95oC 15 sec 55-70oC 60 sec / melt analysis analysis C(t) vs Annealing Temp C(t) vs Annealing Temp 72 72 70 70 68 68 66 66 Annealing Temp Annealing Temp 64 64 62 62 60 60 58 58 56 56 54 54 52 52 25 30 35 40 45 50 55 25 30 35 40 45 50 55 C(q) C(q) CCl26 amplified using Other Reagent B: 5 ul Assay CCl26 amplified using Other Reagent C: 5ul Assay 95oC 20sec / 50x 95oC 3 sec 55-70oC 30 sec / melt analysis 95oC 20sec / 50x 95oC 3 sec 55-70oC 30 sec / melt analysis www.bio-rad.com/genomics/pcrsupport

How did they fare?AMPLIFICATION CCl26 amplified using Bio-Rad SsoFastTM EVAGreen® Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport

Assay ValidationAMPLIFICATION • Assays must be validated to ensure target specificity, dynamic range and sensitivity. • Specificity can be initially established using melt curve analysis but subsequently need to be confirmed using sequencing or another confirmatory tool. • Dynamic range should cover the real life experimental range the assay will cover. • If an assay needs to discriminate small differences, the assay’s capability to do so must be demonstrated. • Additionally, very low copy and detection assays need to be validated using tools such as Poisson distribution analysis. www.bio-rad.com/genomics/pcrsupport

Validation of dynamic range and sensitivityAMPLIFICATION • Confirming dynamic range of an assay is as simple as generating a sequential dilution series and generating a standard curve. • Dynamic range of assay should encompass the range of interest. • There is very little use in having standard curve with a dynamic range spanning 8 orders when all the samples are within 10 fold of one another. www.bio-rad.com/genomics/pcrsupport

Large dynamic rangeAMPLIFICATION 1/10 1/10 1/10 1/10 1/10 1/10 1/10 1/10 Blank 10^9 copies 10^7 copies 10^5 copies 10^3 copies 10 copies 10^8 copies 10^6 copies 10^4 copies 100 copies www.bio-rad.com/genomics/pcrsupport

Large dynamic rangeAMPLIFICATION GAPDH amplified using Bio-Rad SsoFast EVAGreen Supermix: 20ul Assay 98oC 30sec / 50x 95oC 1 sec 60oC 1 sec / melt analysis www.bio-rad.com/genomics/pcrsupport

High sensitivity assayAMPLIFICATION 1/2 1/2 1/2 1/2 1/2 1/2 1/2 1/2 Blank 25 ng / well 6.25 ng / well 1.56 ng / well 390 fg / well 98 fg / well 12.5 ng / well 3.13 ng / well 781 pg / well 195 fg / well www.bio-rad.com/genomics/pcrsupport

High sensitivity assayAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 58oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport

Standard CurveAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 58oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport

qPCR ProtocolAMPLIFICATION www.bio-rad.com/genomics/pcrsupport

qPCR ValidationAMPLIFICATION www.bio-rad.com/genomics/pcrsupport

AMPLIFICATION • Successful assay Design • Conformance with MIQE guidelines • Confidently move forward with experiments www.bio-rad.com/genomics/pcrsupport

Parameters for ConsiderationAMPLIFICATION Sometimes a little additional optimization is required • Primer concentration • 2nd structures on template • AT rich regions • Multiple assays on plate • Amplicon Size • Sequence homology • Inhibitors www.bio-rad.com/genomics/pcrsupport

Primer TitrationAMPLIFICATION • Primer concentration plays an important role in qPCR amplification. • Typical concentrations go from 200nM to 500nM but can vary from 50nM to 800nM and sometimes higher. • High primer concentrations dramatically increase the incidence of non specific amplification and primer-dimers. • Reasonably well designed assays work best at normal primer concentrations www.bio-rad.com/genomics/pcrsupport

100nM each PrimerAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport

100nM each PrimerAMPLIFICATION Replicates Mean C(t) : 27.24 Standard Deviation : 0.284 CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport

300nM each Primer - OptimalAMPLIFICATION Replicates Mean C(t) : 26.54 Standard Deviation : 0.185 CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport

Melt curveAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport

2nd Structures on templateAMPLIFICATION CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT TGACTTGTTC AT Maintain forward primer at 200nM Titer reverse primer www.bio-rad.com/genomics/pcrsupport

200nM forward -- 100nM reverseAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport

200nM forward -- 100nM reverseAMPLIFICATION Replicates Mean C(t) : 35.91 Standard Deviation : 0.540 CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport

2nd Structures on templateAMPLIFICATION • When working with a region of DNA known to have a secondary structure; it can be advantageous to increase the concentration of that primer, all the while maintaining the normal primer at regular levels. • Caution must be used when using high primer concentrations to avoid nonspecific amplifications. • When working with sequences rich in secondary structures, designing primers with higher annealing temperatures, 65oC and above, should be considered as the higher temperatures will help dissociate some of the structures. www.bio-rad.com/genomics/pcrsupport

AT rich sequences on templateAMPLIFICATION CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT TGACTTGTTC AT Maintain forward primer at 200nM Titer reverse primer www.bio-rad.com/genomics/pcrsupport

Running Multiple assays on the same plateAMPLIFICATION • There is often a need to run multiple different assays on the same plate. • Assays should run under optimal conditions; with the proper annealing conditions. • Adjusting primers and conditions can help solve these issues. www.bio-rad.com/genomics/pcrsupport

AMPLIFICATION Different sized primers targeting same amplicon 16 16 30 30 www.bio-rad.com/genomics/pcrsupport

AMPLIFICATION Primer length – 16 bases www.bio-rad.com/genomics/pcrsupport

Running Multiple assays on the same plateAMPLIFICATION • Primer size can affect annealing dynamics. • When annealing range is too low, primer concentration can be increased, or primer size can be increased. • When annealing range is too high, primer size can be reduced. • When increasing primer concentrations, as always specificity for the target must be evaluated. www.bio-rad.com/genomics/pcrsupport

Large ampliconsAMPLIFICATION • Classic qPCR rules dictate that amplification products be between 75 and 200 bp in length. • New “ultra fast” reagents allow much larger amplicons to be used in qPCR. • Extending the size of the amplicon should be considered when trying to circumvent secondary structures, sequence homology and unfavorable regions. • Proper validation is required. www.bio-rad.com/genomics/pcrsupport

Large amplicons – dynamic rangeAMPLIFICATION •B-Actin 1076 pb amplicon from plasmid •109 to 10 copy per well 10 fold dilution 109 copies series •5 ul asay run on CFX384 using Bio- Rad’s SsoFast EVA Green Supermix 10 copies •Protocol : 98oC 3 min 45 x 95oC 1 sec 66oC 5 sec melt curve www.bio-rad.com/genomics/pcrsupport

Large amplicons - sensitivityAMPLIFICATION •B-Actin 1076 pb amplicon from plasmid •105 to 200 copy per well 2 fold dilution series 105 copies •5 ul asay run on CFX384 using Bio- Rad’s SsoFast EVA Green Supermix 200 copies •Protocol : 98oC 3 min 45 x 95oC 1 sec 66oC 5 sec melt curve www.bio-rad.com/genomics/pcrsupport

Sequence HomologyAMPLIFICATION • Designing primers on a region of template sequence homologous to another gene should be avoided if possible. www.bio-rad.com/genomics/pcrsupport

CCL26 with homologous sequencesAMPLIFICATION CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT TGACTTGTTC AT Poor specify is likely outcome www.bio-rad.com/genomics/pcrsupport

CCL26 with homologous sequencesAMPLIFICATION CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT TGACTTGTTC AT Increased specify www.bio-rad.com/genomics/pcrsupport

CCL26 with homologous sequencesAMPLIFICATION CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT TGACTTGTTC AT With very difficult targets www.bio-rad.com/genomics/pcrsupport

Sequence HomologyAMPLIFICATION • Designing primers on a region of template sequence homologous to another gene should be avoided if possible. • When inevitable, a single primer can be designed to anneal on a homologous region for a series of genes. The other primer should be annealing on a clean region or one that has no homology with genes annealed by the first primer. • Multiple primers should be designed and tested. • If a single primer anneals multiple to targets, it will generate a linear amplification of DNA, where as, if both primers anneal, the amplification will be exponential. www.bio-rad.com/genomics/pcrsupport

InhibitorsAMPLIFICATION • PCR although a routine process, is an elegant dance, comprised of a series of complex processes and interactions between enzymes, primers, nucleotides, template DNA and buffer components. • Inhibition can be caused by various chemicals, solvents, ions and peptides (to name a few). • Since their presence is never uniformly distributed in samples, they cannot easily be corrected for in the reaction mix. They should be removed from the sample (as possible), or a supermix that can withstand this inhibitory effect should be used. www.bio-rad.com/genomics/pcrsupport

Blood SerumAMPLIFICATION <2.5 % 10 % CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x95oC 1 sec 60oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport

Blood SerumAMPLIFICATION <0.0098 % 0.039 % <0.0089% 0.039% CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: CCl26 amplified using Other Reagent A: 5ul Assay 5ul Assay 95oC 3 min / 50x 95oC 10 sec 60oC 60 sec / melt 95oC 5min / 50x 95oC 15 sec 60oC 60 sec / melt analysis <0.0089% <0.0089% 0.039% 0.039% CCl26 amplified using Other Reagent B: 5ul Assay CCl26 amplified using Other Reagent C: 5ul Assay 95oC 20sec / 50x 95oC 3 sec 60oC 30 sec / melt analysis 95oC 20sec / 50x 95oC 3 sec 60oC 30 sec / melt analysis www.bio-rad.com/genomics/pcrsupport

Understanding your assayAMPLIFICATION www.bio-rad.com/genomics/pcrsupport

Speed - SsoFastAMPLIFICATION SsoFast EvaGreen Supermix Sso7d from Sulfolobus solfataricus – 7kD, 63 aa. – Thermostable (Tm >90°C) – No sequence preference – Binds to dsDNA (3-6 bp/protein molecule) – Monomeric • Minimal inhibition of PCR by use of EvaGreen • Higher activity • Tolerant to PCR inhibitors www.bio-rad.com/genomics/pcrsupport

ThroughputAMPLIFICATION • The CFX384 real-time PCR detection system brings flexibility and ease of use to researchers performing high-throughput real- time PCR in a 384-well format. • With up to 4-target detection, unsurpassed thermal cycler performance, and powerful, yet easy-to-use software, the CFX384 system has been designed for the way you work. – FAST – shorten the time from experiment setup to results – FRIENDLY – a new standard for ease of use, delivering data you can trust with no maintenance – FLEXIBLE – customize a set up that fits individual laboratory needs www.bio-rad.com/genomics/pcrsupport

ConclusionsAMPLIFICATION • The key to successful qPCR experiments lie with proper design, optimization and validation. • qPCR assay optimization and dynamic range validation require very little time and effort and help guarantee that the results will be reproducible and comparable form experiment to experiment. • Implementation of MIQE guidelines is almost seamless. • If potentially interfering elements are discovered at the design and optimization phases, they can be accounted for and possibly corrected. • Designing good assays does not have to be a “chore”, it can be quite fun! www.bio-rad.com/genomics/pcrsupport

AMPLIFICATION • Thank You! • Questions? www.bio-rad.com/genomics/pcrsupport

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A practical approach to assay design for qPCR

by American Biotechnologist on Aug 01, 2011

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