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5 Essential Tips for Real-time PCR Success

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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.

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
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
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The Azure Cielo 3 and Azure Cielo 6 are for Research Use Only. Not for use in diagnostic procedures.

Copyright © 2022 Azure Biosystems. All rights reserved. The Azure Biosystems logo, Azure Biosystems®, and CieloTM are trademarks of the Company. SYBR® is a registered trademark of Life Technologies Corporation. PrimerQuest® and OligoAnalyzerTM are trademarks of Integrated DNA Technologies. All other trademarks, service marks and trade names appearing in this brochure are the property of their respective owners.


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