Reverse Transcription Protocol (One-step SYBR Green I Dye 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.

Equipment

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

Reagents

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

In the example given below, the primer concentrations can be adjusted according to the results of optimization procedures (see Primer Concentration OptimizationPrimer Optimization Using Temperature Gradient and Assay Optimization and Validation).

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.    Prepare a master mix for each reaction and control requiring RT enzyme plus 10% extra to allow for pipetting    error
       according to Table P11-28.

4.    Prepare a master mix for each control requiring NO RT enzyme plus 10% extra to allow for pipetting error referring to
       Table P11-28 but replacing the enzyme with PCR grade water.

Table P11-28. Reaction Master Mix for One-step SYBR Green I RT-PCR.

 

Reagents Volume (μL) per
Single 25 μL Reaction
2× SYBR Green Quantitative RT-PCR Buffer, Sigma QR0100 12.5
Reference dye (optional) Instrument-specific, see Table P4-7 0.025
Primer F (10 μM) 1.125
Primer R (10 μM) 1.125
PCR grade water 9.1
MMLV RT enzyme 0.125

 

5.    Add 1 μL total RNA (250–2500 ng total per reaction) to each PCR tube. If using a PCR plate, follow a plate schematic to
       ensure that the reaction mix, samples and controls are added to the correct wells.

6.    Add 24 μL master mix to each well, adding the No RT mix to the minus RT control samples.

7.    After sealing each reaction or the plate, vortex gently to mix contents.

8.    Centrifuge briefly to collect components at the bottom of the reaction tubes.

9.    Set the real-time qPCR according to Table P11-29.

Table P11-29. RT PCR Cycling Parameters for One-step SYBR Green I 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 95 5 sec
Step 2 55 15 sec
Step 3 72 10 sec

 

10.    Run post-reaction melt analysis.

Materials