Reverse Transcription Protocol (One-step Probe Detection)
Section Overview
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 (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 (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 oligos.
Supplies
- Sterile filter pipette tips
- Sterile 1.5 mL screw-top microcentrifuge tubes (CLS430909)
- PCR tubes and plates, select one to match desired format:
RT-PCR Method
- Place kit components and RNA samples on ice.
- Mix and then centrifuge briefly to collect contents at the bottom of the tube.
- Refer to Table 1 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.
- Refer to Table 1 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.
*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.
- Add 1 μL RNA (250-2,500 ng) to each reaction tube, replacing with water for No Template Controls.
- 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.
- After sealing each reaction, vortex gently to mix contents. Centrifuge briefly to collect components at the bottom of the reaction tube.
- Set the real time qPCR instrument program according to Table 2.
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