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Reverse Transcription Protocol Using SYBR Green 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 before adding it to the PCR/qPCR, or to prepare a one-step reaction where both processes are performed sequentially.

Equipment

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

Reagents

Supplies

  • Sterile filter pipette tips
  • Sterile 1.5 mL screw-top microcentrifuge tubes (Product No. CLS430909)
    • PCR tubes and plates, select one to match desired format:
    • Individual thin-walled 200 μL PCR tubes (Product No. Z374873 or P3114)
    • Plates
        - 96-well plates (Product No. Z374903)
        - 384-well plates (Product No. Z374911)
    • Plate seals
        - ThermalSeal RTS™ Sealing Films (Product No. Z734438)
        - ThermalSeal RT2RR™ Film (Product No. Z722553)

Method

In the example given below, the primer concentrations can be adjusted according to the results of optimization procedures (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.
  1. 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.
  2. Add 24 μL master mix to each well, adding the No RT mix to the minus RT control samples.
  3. After sealing each reaction or the plate, vortex gently to mix contents.
  4. Centrifuge briefly to collect components at the bottom of the reaction tubes.
  5. 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.
  1. Run post-reaction melt analysis.
Materials
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