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Standard Reverse Transcription Protocol (Two-step)

Reverse Transcription

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.

Equipment

  • Standard PCR instrument or heating block
  • Laminar flow hood for RT set up (optional)

Reagents

  • RNA (stock solution approximately 1 μg/μL).
  • ReadyScript® two-step cDNA synthesis kit (Sigma 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.

Supplies

Method

Preparation

  1. Place ReadyScript® kit components and RNA samples on ice.
  2. Mix and then centrifuge briefly to collect contents at the bottom of the tube.

Reaction

  1. 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.

Table P10-26. ReadyScript® cDNA Synthesis Reaction Master Mix.

 

Reagent Volume (μL) per
Single 20 μL Reaction

ReadyScript® cDNA Synthesis Mix (5× RT blend) 4
Total RNA template—variable (1 μg to 10 pg) 1
PCR grade water 15

 

        2.  After sealing each reaction, vortex gently to mix contents. Centrifuge briefly to collect components at the
             bottom of the reaction tube.

        3.  Incubate reaction mix according to Table P10-27.

Table P10-27. ReadyScript® cDNA Synthesis Reaction Temperature Incubation Profile.

 

Temperature (°C) Time (min)
25 5
42 30
85 5
4 Hold

 

        4.  After completion of cDNA synthesis, use 1:5 to 1:10 of the first-strand reaction (2–4 μL) for PCR amplification.
            Note: when using ReadyScript® reagents, 2–4 μL undiluted cDNA can be added to PCR without causing inhibition.
            If desired, cDNA product can be diluted with 10 mM Tris- HCl (pH 8.0), 0.1 mM EDTA and stored at –20 °C.

Materials