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5 Essential Tips for Real-time PCR Success

Whether preparing for your first qPCR experiment or a seasoned expert, when it comes to perfecting qPCR assays, everyone has room to maximize their success. Test your qPCR knowledge and consider these important tips based on your experience.

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1. DESIGN GREAT PRIMERS AND PROBES

  • Determine your reference gene; great primers may already exist
  • Target amplicon size = 70–150 bp
  • Span an exon-exon junction
  • GC content = 40–60%
  • Tm = 50–65°C
  • Restrict the number of identical nucleotide runs
  • Verify specificity with Primer-BLAST
  • Ensure minimal secondary structures and prevent primer dimer formation with an online tool like IDT’s OligoAnalyzerTM
  • Optimize Ta with a temperature gradient run on a qPCR device
  • Use an online tool like IDT’s PrimerQuest® to design specific primer/probe combinations
  • Store stock oligos (100μM) and diluted aliquots (10–20μM) in nuclease-free H2O at -20°C

2. ISOLATE HIGH-QUALITY RNA

  • RNA is very sensitive to degradation; use PPE, ensure all tools and working area are sterilized with H2O2, and keep samples on ice
  • Ensure high quality RNA (260/280 ratio of 1.8–2.0; ideally 2.0) and quantity (varies) before proceeding to cDNA synthesis
  • If concerned about quality, run RNA out on a gel; smearing as opposed to two clean (2:1) bands indicates poor quality
  • Treat with DNase to remove contamination from genomic DNA (gDNA) prior to cDNA synthesis

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3. REFINE TEMPLATE QUANTITY
& REACTION EFFICIENCY

  • Vortex and quick-spin down all reagents before use, making sure to use a low volume (e.g. P10) pipette when needed
  • Serially dilute template cDNA to identify a dilution that yields ideal cycle threshold (Ct) values of 20–30
  • Verify reaction efficiency [10(-1/slope)-1]*100% and R2 > 0.95 from a standard curve for all primer pairs; always use fresh dilutions
  • Validate multiplexed assays together

4. INCLUDE THE PROPER CONTROLS

  • A no template control (NTC) will determine if contamination is present in your master mix
  • A positive control ensures the reaction conditions are met and helps determine if your new primers/probes work
  • A no reverse transcriptase (RT) control will determine if you have DNA contaminants (e.g. from gDNA)
  • For SYBR® based assays, use a melt curve at the end of cycling to determine that only one product is amplified
  • An internal positive control will determine if PCR inhibitors are present

5. SELECT THE APPROPRIATE
METHOD FOR Ct ANALYSIS:
THRESHOLD VS REGRESSION

  • Verify the cycling conditions are correct (especially if using a shared device)
  • The regression mode of analysis is default on many qPCR devices
  • For threshold analysis, set the baseline to be two cycles earlier than the Ct value for the most abundant sample; the threshold should be set to the product’s exponential growth phase