Reverse Transcription Protocol (One-step Probe Detection)

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.

In some cases it is preferable to measure the target transcript directly, without preparing cDNA from the entire RNA sample. Such situations may include measurements on highly degraded RNA or when there is limiting sample.

Equipment

  • Quantitative PCR instrument
  • Laminar flow hood for PCR set up (optional)

Reagents

  • RNA (Stock, approximately 1 μg/μL)
  • Quantitative RT-PCR Ready Mix (Sigma QR0200)
  • PCR grade water: PCR grade water (W1754 or W4502) as 20 mL aliquots; freeze; use a fresh aliquot for each reaction.
  • Forward and reverse primers concentration stocks (10 μM working stocks are suitable for use in single reactions whereas 100 μM working stocks are suitable for use in multiplex reactions).
  • Specific target detection probes (see PCR/qPCR/dPCR Assay Design) concentrated stocks (10 μM working stocks are suitable for use in single reactions whereas 100 μM working stocks are suitable for use in multiplex reactions).
    •    Custom oligos can be ordered at sigma.com/oligos.
Instrument Final Reference
Dye Concentration
μL of Reference Dye
(per 20 μL Reaction)
Applied Biosystems 5700 0.2
Applied Biosystems 7000 0.2
Applied Biosystems 7300 0.2
Applied Biosystems 7500 0.1× 0.02
Applied Biosystems 7500 Fast 0.1× 0.02
Applied Biosystems 7700 0.2
Applied Biosystems 7900 0.2
Applied Biosystems 7900 HT Fast 0.2
Applied Biosystems 7900HT 0.2
Applied Biosystems StepOnePlus™ 0.2
Applied Biosystems StepOne™ 0.2
Applied Biosystems ViiA 7 0.1× 0.2
Bio-Rad CFX384™ not used -
Bio-Rad CFX96™ not used -
Bio-Rad MiniOpticon™ not used -
Bio-Rad/MJ Chromo4™ not used -
Bio-Rad/MJ Opticon 2 not used -
Bio-Rad/MJ Opticon™ not used -
Cepheid SmartCycler® not used -
Eppendorf Mastercycler® ep realplex not used -
Eppendorf Mastercycler® ep realplex2 s not used -
Illumina Eco qPCR not used -
Qiagen/Corbett Rotor-Gene® 3000 not used -
Qiagen/Corbett Rotor-Gene® 6000 not used -
Qiagen/Corbett Rotor-Gene® Q not used -
Roche LightCycler® 480 not used -
Stratagene Mx3000P® 0.1X 0.02
Stratagene Mx3005P™ 0.1X 0.02
Stratagene Mx4000™ 0.1X 0.02

 

Supplies

Method

1.    Place kit components and RNA samples on ice.

2.    Mix and then centrifuge briefly to collect contents at the bottom of the tube.

3.    Refer to Table P12-30 to prepare a master mix that is sufficient to analyze each sample and controls in duplicate
       plus prepare 10% extra to allow for pipetting error.

4.    Refer to Table P12-30 to prepare a master mix that is sufficient to analyze the NO RT enzyme samples and controls in
       duplicate plus prepare 10% extra to allow for pipetting error, replacing the RT enzyme with PCR grade water.

Table P12-30. Reaction Master Mix for One-step Reverse Transcription, Probe Detection.

Reagent <Volume (μL) per
Single 25 μL Reaction
Quantitative RT-PCR 2X Buffer, Cat. No. QR0200 12.5
Ref dye (optional). Instrument specific, see Table P4-7. 0.025
*Primer F (10 μM) 1.125
*Primer R (10 μM) 1.125
*Probe (10 μM) 0.625
PCR grade water 8.475
MMLV RT enzyme 0.125

*The primer and probe concentrations given are suitable for an entry test of the assay but should be adjusted according to the results of optimization.

 

5.    Add 1 μL RNA (250-2500ng) to each reaction tube, replacing with water for No Template Controls.

6.    Add 24 μL appropriate reaction master mix (from steps 3 and 4) to each well. If using a PCR plate, follow a plate
       schematic to ensure that the reaction mix, samples and controls are added to the correct wells.

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

8.    Set the real time qPCR instrument program according to Table P12-31.

Table P12-31. PCR Conditions for One-step RT-qPCR.

Cycling Conditions Temp (°C) Time
First Strand Synthesis 42-44 30 min
Denaturation/RT inactivation 94 30 sec
Steps 1–3 are repeated through 40 cycles
Step 1 94 5 sec
Step 2 55 15 sec
Step 3 72 10 sec

 

Materials