Ahmed Abdelhamid

You can extract your desired RNA from your samples using RNA extraction kit (QIAamp or RNeasy or any bead based extraction kit) and synthesize cDNA. You may use one of following kits; protocol, reaction volume everything is described in kit manual.

1. High-Capacity cDNA Reverse Transcription Kit

https://lusis.genetics.ucla.edu/sites/default/files/ABI-High-Capacity-cDNA-Synthesis.pdf

2. LunaScript®  RT SuperMix Kit: https://international.neb.com/protocols/2018/01/31/protocol-for-lunascript-rt-supermix-kit-e3010

Try to check the quality of your isolated RNA on an agarose gel if it is in good condition before cDNA synthesis. Also use DNase I before cDNA synthesis and perform control PCR on your cDNA along with the RT negative control (RNA without reverse transcriptase enzyme), the negative control without RNA teplate (use water instead of RNA) and the positive control.

We use Qiagen RNeasy isolation kits, DNase from Thermo and RevertAid cDNA synt. kit also by Thermo.

First and foremost step is to isolate RNA carefully, that is without loosing its integrity. It can be done using TRIzol method or commercially available kits.

Now, you can synthesize the first strand of cDNA using commercially available kits as mentioned below:

Verso cDNA kit: https://www.thermofisher.com/order/catalog/product/AB1453A

Quantitect Reverse transcription kit: https://www.qiagen.com/us/products/discovery-and-translational-research/pcr-qpcr-dpcr/real-time-pcr-enzymes-and-kits/reverse-transcription-cdna-synthesis-qpcr/quantitect-reverse-transcription-kit/