Multiplex qPCR Protocol

Quantitative PCR Protocols

Although quantitative PCR uses the same basic concept as traditional PCR, the reactions differ in that the amplicons are generally smaller and are detected indirectly using an additional dye or labeled probe or primer.

In the PCR Technologies Guide, the requisite components and quality control requirements for qPCR experiments were described in detail. With those in mind, the following is a protocol that can be used as a basic template for qPCR incorporating up to four detection probes. In these reactions, primers and probe are included at a final concentration of 200 nM and are run using LuminoCt® ReadyMix™ (Sigma). However multiplex experiments require optimization and it is advisable to test the assay combinations by adding each to the multiplex sequentially. A detailed assay optimization protocol for each single assay is described in Primer Concentration Optimization and Primer Optimization Using Temperature Gradient.


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



1.    Defrost all reaction components on ice, taking care to protect the probes from exposure to light.

2.    Calculate the number of reactions required to enable samples and controls to be run in duplicate. Include two
       No Template Controls (NTC).

3.    Prepare a probe blend and a primer blend using Tables P6‑13A and 13B as a guide. For reactions
       requiring 2 or 3 probes, adjust to the total volume (with PCR grade water). After optimizing primer
       concentration, adjust primer concentrations/volumes accordingly. The volumes stated in Tables P6‑13A
       and 13B are sufficient to run 250 reactions, scale up accordingly for more reactions.

Table P6-13A. Volume Of Stock Probe to Blend for 200 nM Final Concentration in Each Reaction. Amend volumes in the blend following optimization.

Probe (100 μM) Volume (μL)
Target 1 10
Target 2 10
Target 3 10
Target 4
PCR grade water 60


Table P6-13B. Volume of Stock Primers to Blend for 200 nM Final Concentration in Each Reaction. Amend volumes in the blend following optimization.

Forward Primer
(100 μM)
Reverse Primer
(100 μM)
Target 1 10 Target 1 10
Target 2
10 Target 2
Target 3
10 Target 3
Target 4
10 Target 4
PCR grade water     20


4.    Prepare a master mix for all reactions according to Table P6‑14 (calculate volumes for each reaction and add 10% to
       allow for pipetting error). Mix well, avoiding bubbles.

Table P6-14. Master Mix for Mulitplex Probe qPCR.

Reagent Volume (μL) per
Single 20 μL Reaction
2× LuminoCt qPCR ReadyMix 10
Probe Mix (Table P6-13A) 0.4
Primer mix (Table P6-13B) 0.4
PCR grade water 4.2


5.    Add 5 μL of water to the NTC reaction tubes.

6.    Add 5 μL of cDNA/gDNA solution to the appropriate tubes/wells.

7.    Aliquot 15 μL template master mix remaining from step 4 into the PCR tubes.

8.    Cap tubes or seal the PCR plate and label (according to instrument requirements). (Make sure the labeling
       does not obscure instrument excitation/detection light path.)

9.    Centrifuge briefly and visually check that all tubes/wells contain sample at the bottom at the correct volume.

10.   Run samples according to the two-step protocol (Table P6‑15), repeating steps 1–2 through 40 cycles.
        (Note: These conditions are specific for FAST cycling protocols).

11.   For reactions containing Scorpions® Probes or Molecular Beacons, a three-step protocol (Table P6‑16) may
        result in more efficient/sensitive detection. When adopting this protocol, the annealing temperature of step 2
        can be optimized. Cycle steps 1–3 through 40 cycles.

Table P6-15. Fast PCR Cycling Conditions.

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


Table P6-16. Three-step PCR Cycling Conditions for use with Scorpions® Probes or Molecular Beacons.

3-Step Cycling Conditions (for Use with Scorpions®
Probes or Molecular Beacons)
Temp (°C) Time (sec)
Initial denaturation 95 30
Steps 1–3 are repeated through 40 cycles
Step 1 Denature 95 5
Step 2 Anneal (select optimized Ta) 58 15
Step 3 Extend 72 10