1-step qRT-PCR Protocol for LightCycler® 480
This protocol is intended for use with the Roche LightCycler® 480 real-time PCR instrument.
Protocole KAPA™ SYBR® FAST One-Step qRT-PCR Kit Optimized for Roche LightCycler® 480
Réactifs :
- Template RNA
- Forward primer
- Reverse primer
- KAPA SYBR® FAST qPCR Master Mix (2X) with ROX Reference Dye
- 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), template RNA and primers. ROX reference dye is included in the Master Mix at a final concentration of 500 nM.
- 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 - 5 μl 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 | 20 μl rxn | |
| Nuclease-free water up to 20 μl | As required | |
| KAPA SYBR® FAST qPCR Master Mix (2X) | 1X | 10 μl |
| Forward Primer (10 μM) | 200 nM | 0.4 μl |
| Reverse Primer (10 μM) | 200 nM | 0.4 μl |
| dUTP (10 mM) (optional) | 200 μM | 0.4 μl |
| KAPA RT Mix (50X) | 1X | 0.4 μl |
| Template RNA | variable | < 5 μl |
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 20 μl to 10 μl if low volume tubes/plates are used.
- Cap or seal the reaction tube/plate and centrifuge briefly.
Step 3: Run the qPCR Reaction
- Program the following cycling protocol:
| Program name | Cycle | Analysis mode | |
| Reverse Transcription | 1 | None | |
| Amplification | 40 (1) | Quantification | |
| Melting Curve | 1 | Melting curves | |
| Cooling | 1 | None | |
| Program Name | Target (ºC) | Acquisition Mode | Hold (hh:mm:ss) |
| Reverse Transcription | 42°C | None | 00:05:00 |
| 95°C | None | 00:03:00 (2) | |
| Amplification | 95°C | None | 00:00:10 |
| Primer dependent (3) | None | 00:00:20 (4) | |
| 72°C | Single | 00:00:01 (5) | |
| Melting Curve | 95°C | None | 00:00:05 |
| 65°C | None | 00:01:00 | |
| 97°C | Continuous | 5 - 10 Acquisitions/°C | |
| Cooling | 40°C | None | 00:00:10 |
(1) 40 cycles are suitable for most assays, however this may be reduced depending on initial target concentration.
(2) 20 sec at 95 °C is sufficient time for DNA polymerase activation, however 3 min is required to fully inactivate the reverse transcriptase prior to cycling.
(3) qPCR primers are typically designed for optimal annealing at 60 °C, however optimal annealing temperatures may differ from calculated values.
(4) It is not recommended to use less than 20 sec for primer annealing.
(5) Due to the high processivity of the engineered KAPA SYBR® DNA Polymerase, only 1 sec at 72 °C is sufficient time for extension of amplicons <400 bp.
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