Universal protocol of 1-step quantitative RT-PCR

Protocole universel KAPA™ SYBR® FAST One-Step qPCR




Réactifs :
- Template RNA
- Forward primer
- Reverse primer
- KAPA SYBR® FAST qPCR Master Mix (2X)
- ROX Reference Dye Low/High (50X)
- dUTP
- KAPA RT Mix (50X)


Protocole général de qPCR

Step 1: qPCR Reaction Setup
- Before preparing qRT-PCR reactions, thoroughly mix the KAPA SYBR® FAST qPCR Master Mix (2X), KAPA RT Mix (50X), dUTP (10 mM), ROX Reference Dye High/Low, template RNA and primers.
- Keep all kit components and assemble all reactions on ice to avoid premature cDNA synthesis.
- The recommended RNA input is from 1 pg to 100 ng total RNA.
- Prepare a reaction cocktail to reduce pipetting errors. Dispense equal aliquots into reaction tubes. Add RNA to each reaction as a final step. Addition of 2 - 5μl volumes of RNA will improve assay precision.
- Include a No Template Control (NTC) and No RT Control (NRT) when necessary. The NTC will enable detection of contamination in the reaction components. The NRT control tests for contaminating genomic DNA in the reaction.
- Calculate the required volumes of each component based on the following table:

	Final concentration	20μl rxn
Nuclease-free water up to 20μl		As required
KAPA SYBR® FAST qPCR Master Mix (2X)	1X	10μl
Forward Primer (10μM)	200 nM	0.4μl
Reverse Primer (10μM)	200 nM	0.4μl
dUTP (10 mM) (optional)	200μM	0.4μl
ROX High/Low (optional)		0.4μl
KAPA RT Mix (50X)	1X	0.4μl
Template RNA	variable	< 5μl

Step 2: Plate Setup
- Transfer the appropriate volume of reaction mixture to each well of a PCR tube/plate. Reaction volumes may be scaled down from 20μl to 10μl if low volume tubes/plates are used.
- Cap or seal the reaction tube/plate and centrifuge briefly.

Step 3: Run the qPCR Reaction
- If applicable, select fast mode on the instrument.
- Program the following cycling protocol:

Step	Temperature	Duration	Cycle
cDNA synthesis	42°C	5 min	Hold
Inactivate RT	95°C	2-5 min	Hold
Denature	95°C	3 sec	40
Anneal/extend	60°C	≥ 20 sec	40
Dissociation	Accordind to	instrument	guidelines

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