AAT Bioquest

What are the factors I should consider to get accurate and reproducible results for my probe-based qPCR?

Posted May 12, 2023


Consider these 10 factors to get accurate and reproducible results for your probe-based qPCR

  1. Use high quality RNA that is prepared from fresh tissue or from tissue treated with an RNA stabilization solution. Impure or degraded RNA can compromise the efficiency of the reaction and reduce yield.
  2. Use good primer design software that includes adjustable parameters for optimal primer and probe design. 
  3. Use a master mix that contains all the reagents necessary for qPCR. This will improve reproducibility by minimizing sample-to-sample and well-to-well variations. Choosing a master mix that contains a passive reference dye such as ROX (6-carboxyl-X-Rhodamine) will further reduce well-to-well variation. 
  4. Include a ‘No Amplification Control’ or minus-reverse transcriptase control in your experiment to detect the presence of contaminating DNA in the sample. 
  5. Take care to set the baseline properly to obtain accurate Ct (cycle threshold) values. The correct way is to set the baseline 2 cycles earlier than the Ct value for the most abundant sample. 
  6. Perform dissociation or melting curves when using SYBR Green. Ideally, the sample should yield a sharp peak at the melting temperature of the amplicon. A series of peaks indicates that there is insufficient discrimination between specific and non-specific reaction products. 
  7. Include an invariant endogenous control in the assay to correct for sample-to-sample variations and improve the reliability of your qPCR experiment. 
  8. Ideally, the efficiency of the PCR should be 90 - 110% (3.6 > slope > 3.1). Confirm the efficiency of the reaction using the formula Eff =10 (-1/slope) -1. If the result falls outside of the efficiency range, either optimize the qPCR further or design alternative amplicons. 
  9. Prepare standard curves for each gene under study for absolute or relative RNA quantitation. The standard curve should extend above and below the expected abundance of your target. To differentiate between specific and non-specific products include additional input quantities such as the minimum and maximum RNA amounts above and below the limit of detection. 
  10. Decontaminate all surfaces in the PCR area regularly using a DNA decontamination solution to prevent cross-contamination. 
Additional resources

qPCR and qRT-PCR analysis: Regulatory points to consider when conducting biodistribution and vector shedding studies

Real-Time PCR (qPCR)

ROX Reference Dye *50X fluorescence reference solution for PCR reactions*