qPCR Gene Expression/Copy Number/SNP Analysis Using Probe Detection Protocol

Optimization of qPCR Conditions

Optimization of qPCR conditions is important for the development of a robust assay. Indications of poor optimization are a lack of reproducibility between replicates as well as inefficient and insensitive assays. The two main approaches are optimization of primer concentration and/or annealing temperatures.

The most common application for qPCR is the measurement of a gene transcript or copy number quantity relative to one or more reference genes using probe detection. The reactions may be designed such that each target is detected as a single reaction (simplex) or such that all probes can be added to a single reaction to detect multiple targets simultaneously (multiplex). It is essential that multiplex assays are designed to be compatible with each other (see PCR/qPCR/dPCR Assay Design). When using probes for SNP or mutation analysis both probes, i.e. specific to mutant and wild type targets are added to the reaction along with a single set of primers.

After optimization of the assays for both target and reference/control genes, these are used to measure the quantity of target. A ratio is determined between the quantity of gene of interest (GOI) and the reference gene as described in Data Analysis.

In this protocol, the probe is included at 250 nM and the primers at 450 nM. However, it is advisable to optimize these conditions and adjust the reaction volumes accordingly. For a multiplex reaction, add all primers and probes to the reaction mix. It may be necessary to use a more concentrated stock for oligos or prepare a concentrated oligo blend (see Multiplex qPCR).


  • Quantitative PCR instrument
  • Laminar flow hood for PCR set up (optional)


  • cDNA/gDNA undiluted for standard curve.
  • Sample cDNA diluted 1:10, gDNA 10 ng per analysis.
  • LuminoCt qPCR ReadyMix (Sigma L6669).
  • PCR grade water: PCR grade water (W1754 or W4502) as 20 mL aliquots; freeze; use a fresh aliquot for each reaction.
  • Forward and reverse primers for test and reference genes (stock 10 μM).
  • Probe for each target (stock 10 μM) using different fluorescent labels for different targets (e.g., FAM for GOI and
    HEX/JOE for REF).


Notes for this Protocol

  • cDNA is generated using random priming or oligo-dT method (see Standard Reverse Transcription Protocol (Two-step)).
  • If using a PCR plate, follow a plate schematic to ensure that the reaction mix, samples and controls are added to the correct wells.
  • All tests are run as duplicate reactions.
  • The protocol shows detection of 2 genes (e.g., GOI and ref gene).


1.    Prepare a different qPCR master mix for each primer pair to be analyzed. Calculate sufficient mix for the standard curve
       reactions (described as six dilutions below), No Template Controls (NTC) and for each sample, all in duplicate plus an
       extra 10% to allow for pipetting errors (Table P18-45).

       Note: Adjust water volume to accommodate addition of additional primers and probes for multiplex reactions or add
       blended, concentrated stocks (see Multiplex qPCR).

Table P18-45. Reaction Master Mix for Probe Detection.


Reagents Volume (μL) per Single
Reaction 25 μL
2× LuminoCt qPCR ReadyMix 12.5
Forward primer GOI (10 μM) 1.125
Reverse primer GOI (10 μM) 1.125
Forward primer REF (10 μM) 1.125
Reverse primer REF (10 μM) 1.125
Probe GOI (10 μM) 0.625
Probe REF (10 μM) 0.625
PCR grade water 1.75


2.    Prepare a 1:10-fold dilution (or suitable dilution) of suitable standard curve template so that there is 20 μL of each
       dilution (six dilutions in total).

3.    Add 5 μL of appropriate template serial dilution (standard curve), template, controls template or water (NTC) to the
       defined tubes or wells.

4.    Add 20 μL of master mix to each tube/well. 5. Cap tubes or seal the PCR plate and label. (Make sure the labeling does
       not obscure the instrument excitation/detection light path.)

6.    Run samples according to Table P18-46 ensuring that data are collected for all probe labels.

7.    See Data Analysis.

Table P18-46. PCR Cycling Conditions for Muliplex Target Quantification.


Cycling Conditions Temp (°C) Time (sec)
Denaturation/Hot Start 95 30
Steps 1–2 are repeated through 40 cycles
Step 1 95 5
Step 2 60 30