Reverse transcription (RT) is the process of converting RNA to cDNA using a reverse transcriptase enzyme and dNTPs.
The RT step may be performed on total RNA such that a global cDNA is produced that is representative of all of the RNA transcripts in the sample (usually via a two-step protocol), or in a gene-specific approach such that only the RNA of interest is converted to cDNA (usually following a one-step protocol).
The following experiments can be used as basic RT protocols that can be modified to suit particular requirements. It is customary to either prepare cDNA using a two-step process with subsequent dilution of the cDNA prior to adding it to the PCR/qPCR, or to prepare a one-step reaction where both processes are carried out sequentially.
- Standard PCR instrument or heating block
- Laminar flow hood for RT set up (optional)
- RNA (stock solution approximately 1 μg/μL).
- ReadyScript® two-step cDNA synthesis kit (RDRT). Alternative reverse transcription kits can also be used in conjunction with oligo-dT primers and/or random primers.
- PCR grade water: PCR grade water (W1754 or W4502) as 20 mL aliquots; freeze; use a fresh aliquot for each reaction.
- Sterile filter pipette tips
- Sterile 1.5 mL screw-top microcentrifuge tubes (CLS430909)
- PCR tubes and plates, select one to match desired format:
• Individual thin-walled 200 μL PCR tubes (Z374873 or P3114)
- 96-well plates (Z374903)
- 384-well plates (Z374911)
• Plate seals or caps
- ThermalSeal RTS™ Sealing Films (Z734438)
- ThermalSeal RT2RR™ Film (Z722553)
- Place ReadyScript® kit components and RNA samples on ice.
- Mix and then centrifuge briefly to collect contents at the bottom of the tube.
- Determine the number of reactions required, including controls. Calculate the volumes of each component required for all reactions (allow 10% extra for pipetting errors) and combine reagents according to Table P10-26 using 0.2 mL tubes or a 96-well plate sitting on ice. If using a PCR plate, follow a plate schematic to ensure that the reagents are added to the correct wells.