[Skip to Content](https://www.sigmaaldrich.com#main-content) [](https://www.sigmaaldrich.com/US/en) Products Cart0 USEN Products [Login / Register](https://www.sigmaaldrich.com/oidc-sign-in) [Order Lookup](https://www.sigmaaldrich.com/US/en/order-lookup) [Quick Order](https://www.sigmaaldrich.com/US/en/quick-order) Cart0 [Home](https://www.sigmaaldrich.com/US/en)[qPCR](https://www.sigmaaldrich.com/US/en/applications/genomics/qpcr)Multiplex qPCR Protocol # Multiplex qPCR Protocol PCR Technologies Protocols Table of Contents ## 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. 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. ## Equipment - Quantitative PCR instrument - Microcentrifuge - Laminar flow hood for PCR set up (optional) ## Supplies - DNA/cDNA template: cDNA reaction diluted 1:10 to detect a medium to highly expressed targets or 1:2 to 1:5 for rare transcripts. 10 ng to 100 ng gDNA. - LuminoCt® ReadyMix™ ([L6669](https://www.sigmaaldrich.com/US/en/product/sigma/L6669)). - PCR grade water: PCR grade water ([W1754](https://www.sigmaaldrich.com/US/en/product/sigma/W1754) or [W4502](https://www.sigmaaldrich.com/US/en/product/sigma/w4502)) as 20 mL aliquots; freeze; use a fresh aliquot for each reaction. - Forward and reverse primers concentration stocks (100 μM working stocks are suitable for use in multiplex reactions). - Specific target detection probes (see Chapter 6) concentration stocks (100 μM working stocks are suitable for use in multiplex reactions). • Custom oligos can be ordered here. - Sterile filter [pipette tips](https://www.sigmaaldrich.com/US/en/products/labware/liquid-handling-and-dispensing/pipette-tips) - Sterile 1.5 mL screw-top microcentrifuge tubes ([CLS430909](https://www.sigmaaldrich.com/US/en/product/sigma/CLS430909)) - PCR tubes and plates, select one to match desired format: • Individual thin-walled 200 μL PCR tubes ([Z374873](https://www.sigmaaldrich.com/US/en/product/sigma/Z374873) or [P3114](https://www.sigmaaldrich.com/US/en/product/sigma/P3114)) • Plates     - 96-well plates ([Z374903](https://www.sigmaaldrich.com/US/en/product/sigma/Z374903))     - 384-well plates ([Z374911](https://www.sigmaaldrich.com/US/en/product/sigma/Z374911)) • Plate seals     - ThermalSeal RTS™ Sealing Films ([Z734438](https://www.sigmaaldrich.com/US/en/product/sigma/Z734438))     - ThermalSeal RT2RR™ Film ([Z722553](https://www.sigmaaldrich.com/US/en/product/sigma/Z722553)) ## Method 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. | | | |-----------------|-------------| | Probe (100 μM) | Volume (μL) | | Target 1 | 10 | | Target 2 | 10 | | Target 3 | 10 | | Target 4 | 10 | | PCR grade water | 60 | Table P6-13AVolume Of Stock Probe to Blend for 200 nM Final Concentration in Each Reaction. Amend volumes in the blend following optimization. Forward Primer (100 μM)Volume (μL)Reverse Primer (100 μM)Volume (μL) Target 110Target 110 Target 2 10Target 2 10 Target 3 10Target 3 10 Target 4 10Target 4 10 PCR grade water  20 Table P6-13BVolume of Stock Primers to Blend for 200 nM Final Concentration in Each Reaction. Amend volumes in the blend following optimization. 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. ReagentVolume (μL) per Single 20 μL Reaction 2× LuminoCt qPCR ReadyMix10 Probe Mix (Table P6-13A)0.4 Primer mix (Table P6-13B)0.4 PCR grade water4.2 Table P6-14Master Mix for Mulitplex Probe qPCR. 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. | | | | |------------------------------------------|-----------|------------| | 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 P6-15Fast PCR Cycling Conditions. 3-Step Cycling Conditions (for Use with Scorpions® Probes or Molecular Beacons)Temp (°C)Time (sec) Initial denaturation9530 __Steps 1–3 are repeated through 40 cycles__ Step 1 Denature955 Step 2 Anneal (select optimized Ta)5815 Step 3 Extend7210 Table P6-16Three-step PCR Cycling Conditions for use with Scorpions® Probes or Molecular Beacons. ## Materials Sorry, an unexpected error has occurred Response not successful: Received status code 500 __Related Articles__ - [FFPE Tissue](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/dna-and-rna-purification/wga1-ffpe) - [MystiCq® MicroRNA® Quantitation System](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/gene-expression-and-silencing/mysticq-microrna-analysis) - [Multiplex Real-Time PCR](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/pcr/kapa-multiplex-rtpcr) - [Protocol for Conjugating NHS-Ester Modifications to Amino-Labeled Oligonucleotides](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/pcr/nhs-ester-oligonucleotide-conjugation) - [Reverse Transcription Protocol (One-step Probe Detection)](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/pcr/reverse-transcription-one-step-probe-detection) - [The 3'/5' Assay for Analysis of RNA Integrity Protocol](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/qpcr/3-5-assay-for-analysis-of-rna-integrity) - [ChIP-qPCR Data Analysis](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/qpcr/chip-qpcr-data-analysis) - [dPCR Protocol](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/qpcr/dpcr) - [View More](https://www.sigmaaldrich.com/US/en/search/facet-search?focus=sitecontent&term=facet-search) __Related Product Categories__ - [Sealing Films, Foils & Tapes](https://www.sigmaaldrich.com/US/en/products/labware/sealing-films-foils-and-tapes) - [Pipette Tips](https://www.sigmaaldrich.com/US/en/products/labware/liquid-handling-and-dispensing/pipette-tips) Top __Sign In To Continue__ To continue reading please sign in or create an account. 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