220.127.116.11. Two-Step RT-qPCR
The following is a robust, standard qPCR protocol for amplifying and quantifying cDNA templates <300bp in length. The protocol is based on SYBR-green detection chemistry, with modifications for probe-based detection and qualitative PCR indicated:
1.1 3 μl cDNA (pre-diluted 1/10, in nuclease-free water),
1.2. 0.6 µl 10 μM Forward primer (0.3 μM final concentration),
1.3. 0.6 µl 10 μM Reverse primer (0.3 μM final concentration),
[1.4. 0.4 µl* 10 μM TaqMan™ probe* (0.2 μM final concentration*)],
1.5. x µl* TwoStep Buffer* + dNTP (0.2 mM final dNTP),
1.6. y µl nuclease-free water,
1.7. z µl Thermostable DNA polymerase mix,
1.8. 20 µl total volume.
* Use appropriate buffer for either SYBR-green or TaqMan™ probe assays. dNTP is usually included in pre-optimized buffers. If not, add separately to 0.2 mM final concentration.
2. Incubate in real-time thermocycler:
2.1. 5 min:95°C,
2.2. 35 cycles [10 sec:95°C - 30 sec:58°C - read].
3. For SYBR-green assays, follow with Melting Curve analysis:
3.1. 1 min:95°C,
3.2. 1 min:55°C,
3.3. 5 sec:0.5°C:read from 55oC to 95oC.
4. For qualitative PCR, a conventional thermocycler can be used and the products can be analysed by gel, capillary or chip-based electrophoresis.