technical tips for qpcr

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Sample and Experimental Considerations


  • Integrated DNA Technologies Technical Tips for qPCR Sample and Experimental Considerations Aurita Menezes, PhD qPCR Product Manager
  • 1 Session Outcomes We will discuss: Intercalating Dyes (SYBR Green) vs 5 nuclease assays Steps to a Successful qPCR Experiment Assay design criteria Experimental design considerations Sample isolation Sample quantification cDNA synthesis Dye and instrument compatibility Experimental layout Multiplexing Experimental plate layout Methods of quantification
  • 2 5 Nuclease Assays vs Intercalating Dyes (SYBR Green) For use with intercalating dyes such as SYBR Green Primers and probe for 5 nuclease assays
  • 3 Intercalating Dyes (e.g., SYBR Green) cheap nonspecific PCR products and primer-dimers will also contribute to the fluorescent signal longer amplicons create a stronger signal requires melting point curve determination Cannot multiplex or genotype 5 Nuclease assays 3rd sequence in assay (the probe) adds specificity Splice form specific amplification Rare transcript detection Pathogen detection No need for post run analysis such as melt curves Multiple dye ratio options for multiplexing Can perform SNP genotyping Can be slightly more expensive (IDT solution is the PrimeTime Mini) 5 Nuclease Assays vs Intercalating Dyes (SYBR Green)
  • 4 Steps to a Successful qPCR Experiment Assay design RNA cDNA Reverse Transcription qPCR reaction set upAnalysis of data Experimental set-up RNA, DNA isolate, purify, quantify
  • 5 Assay Design
  • 6 Assay Design: Steps to Designing a Good Assay Know your gene How many transcripts are associated with that gene? Which exons are common or specific between the transcripts? Obtain a Refseq accession number Use NCBI databases to identify exon junctions, splice variants, SNP locations Align related sequences For splice specific designs Identify unique regions within which to design primers and probe Blast primer and probe sequences ensure no cross reactivity with other genes within the species
  • 7 Primer and Probe Design Criteria Primer Primer Tm values should be similar +/- 2oC For 5 nuclease qPCR assay, this is normally around 6062oC Aim for 1830 bases Do not contain runs of 4 or more Gs GC content range of 3565% ( ideal 50%) Probe Tm value 410oC higher than primers No runs of consecutive Gs, G+C content 3080% No G at the 5 end Probe length no longer than 30 bases (IDTs ZEN Double Quenched Probes are an exception) Probe can be designed on either the sense or antisense strand Amplicon Size is between 70200 bp If using SYBR Green then amplicon length is designed to be slightly bigger to enable differentiation from primer dimers on a melt curve -> Always BLAST potential primer sequences and redesign if primer sequence cross reacts
  • 8 April 2008 15M SNPs Sept 2010 30M SNPs June 2012 53M SNPs Designed to Avoid SNPs Next Generation Sequencing has significantly increased the number of SNPs and splice variants identified Having up-to-date sequence information is critical to qPCR assay performance
  • 9 The shift due to a SNP at the 3 end of a primer varies from 0 to >10 Cqs. This shift misrepresents a gene expression fold change of as much as 1000 fold! Primers on SNPs Can Lead to Erroneous Gene Expression Data Effect of SNPs within primer locations on Tm
  • 10 PrimeTime Predesigned qPCR Assays for Human, Mouse, and Rat 1. Designed to avoid SNPS 2. We share primer and probe sequences upon purchase 3. Cross reactivity check to eliminate non-specific amplification 4. Reduce impact from secondary structure formation
  • 11 Experimental Design Considerations
  • 12 Experimental Design Considerations Number of reactions Number of replicates Number of samples Number of controls Number of reference genes Sample maximization versus gene maximization
  • 13 Experimental Setup 24h 48h 72h 24h 48h 72h qPCR for 1) gene of interest and 2) Multiple reference sequences tested for stable expression across experimental conditions Normal Mutant Multiple Biological Replicates 2 RT Preps for each sample + 1 No RT Control 3 Technical Replicates for each sample 3 No Template Control for each qPCR assay tested
  • 14 RNA Sample Isolation Guanidinium thiocyanate/phenol:chloroform Pros: Higher yield Works with larger amounts of cells Works better with troublesome tissues (e.g., adipose tissue, bone, cartilage, etc.) Cons: Higher DNA contamination Separate DNase I digestion with additional purification needed Residual phenol inhibits PCR Spin columns are available that have on column DNase digestion yielding Loading capacity maybe limited and small RNA is lost
  • 15 Sample Quantification Many quantification methods are available Spectrophotometry (UV spec or Nanodrop [>2 ng]) Easy to use, high amount of starting material (photometer), not specific for DNA or RNA, highly variable, dont trust absorptions 50 pg/L], BioRads Experion These methods provide a quantitative assessment of the general state of the RNA sample (RIN number) Fluorescent dye detection RNA dyes such as RiboGreen Dye Very sensitive (0.5 ng1 g), expensive Specific for RNA (RiboGreen Dye), dsDNA (PicoGreen Dye)
  • 16 Sample Quantification Always use the same method of quantification Comparison of data obtained using RNA isolated by different methods is not advisable Comparison of data obtained using different RT priming strategies is not recommended Accurate quantification is crucial for true estimation by qPCR
  • 17 Reverse Transcription Reverse transcription can be a major source of error in qRT-PCR RT is a non-linear process: Standardize your input amount Use same amount of RNA (or same number of cells) for all samples RT reagents are inhibitory to PCR, so dilute the reaction
  • 18 Priming Strategy Can Make a Difference Oligo(dT) < Hexamer < Oligo(dT) + Hexamer < Gene Specific Primer Random primers and oligo (dT) primers will produce random cDNA, while gene-specific primers will produce cDNA only for a specific target Random primers Bind to RNA at a variety of complementary sites, resulting in short, partial-length cDNAs Can be used when the template has extensive secondary structure 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 Advantage: Transcriptome is preserved so that any remaining cDNA can be used in other qPCR assays Disadvantage: Low abundance messages may be under-represented due to consumption of reagents during cDNA synthesis of the more prevalent RNAs 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 Gene-specific oligonucleotide primers, which selectively prime the mRNA of interest Yields the least complex cDNA mixture and avoids reagent depletion Gene specific primers can yield earlier Cqs, however only one gene can be tested per cDNA sample Disadvantage: cDNA produced cannot be used for assaying other genes
  • 19 Two -Step Protocol One-Step Protocol Primers used in RT Oligo(dT) primers Random hexamers Gene-specific primers A mix of these Gene-specific primers Advantages Choice of primers Optimize reactions for maximum yield Modulate amount of RT that goes into PCRcontrolling for target abundance Perform multiple PCR reactions on the same cDNA sample Experiment with different RT and Taq enzymes Quick setup and limited handling Easy processing of multiple samples for repetitive tests, or high-throughput screening Fewer pipetting steps, reducing potential errors Eliminates possibility of contamination between the RT and qPCR steps Considerations Requires more setup, hands-on, and machine time Additional pipetting increases the chances for experimental errors and contamination Uses more reagents Must start over, or save RNA aliquot and perform new RT to analyze new target(s) or repeat amplifications Reaction conditions are not optimalefficiency and thus quantification are affected Best for: Amplifying multiple targets from a single RNA source When you plan to reuse cDNA for additional amplifications Working with multiple RNA samples to amplify only a few targets Assays performed repeatedly Choosing Between One-Step and Two-Step RT-qPCR
  • 20 Controls Negative Controls No Template Control (detects contamination) Minus RT (examines genomic DNA presence) Biological Control sample wherein the GOI is not expressed Posit

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