RT-PCR

INTRODUCTION

Reverse Transcription Polymerase Chain Reaction (RT-PCR or rtPCR), not to be confused with real-time PCR (which is also known as quantitative real-time PCR or qPCR), is a technique used in molecular biology to convert mRNA into DNA, which can be used as starting material for PCR. The term reverse transcription derives from the fact that transcription in living eukaryotic cells is the process by which DNA in the nucleus is ‘read.’ This reading process creates mRNA (messenger RNA) which is transported out of the nucleus into the cytoplasm where it is translated into proteins. This is the process of gene expression. In the case of RT-PCR we are reversing this natural process to turn the mRNA we have extracted from our sample cells back into DNA, hence reverse transcription PCR or (RT-PCR or rtPCR).

RT-PCR is also commonly combined with (quantitative) qPCR in order to determine quantitatively the level of gene expression in a sample of cells. By lysing the cells, extracting the RNA and converting it back into DNA via RT-PCR,  the level of mRNA in the two tissues types (for instance cancerous vs normal tissue) can be quantitated.

However, RT-PCR can also be used as a technique on its own to characterize in absolute (non-quantitative) terms the presence of a particular mRNA sequence in a sample.

MATERIALS NEEDED

•  CoolBox 2XT PCR ice-free workstation or, alternatively,  CoolRack M15 and CoolRack PCR modules with ice pan

•  Vortexer

•  Microcentrifuge

•  Heating block for microcentrifuge tubes

•  PCR thermocycler

•  TruCool (RNase-free) microfuge tubes

INSTRUCTIONS

Preparation: Extract RNA from samples (refer to RNA Extraction/Isolation protocol here for instructions).

NOTE: It is crucial to note that mRNA is significantly less thermostable than DNA, and that when working with mRNA, extreme care should be taken in handling the reactions.

Part 1. Reverse Transcription Reaction

Create a master mix of reagents. This should be carried out at <4°C ideally in an ice-free CoolBox XT or on ice with a CoolRack M15 as reverse transcriptase enzymes typically exhibit residual activity at temperatures above 4°C that can result in non-specific background amplfication. The volumes below are per RNA sample, e.g., for 10 samples multiply each component by 10, plus a small excess. Once you have added all of the ingredients together, vortex gently to mix, and return to 4°C while you prepare the RNA samples.

1.1.         3.0μL 10x PCR buffer

1.2.         2.5μL 10mM dNTPs

1.3.         6.0μL 25mM MgCl2

1.4.         1.0μL random primers (1.0μg)

1.5.         17μL distilled (nuclease-free) H2O

1.6.         0.5μL reverse transcriptase enzyme

For each RNA sample combine 5μg RNA with 10μL distilled H2O, in a 1.5ml microfuge tube, vortex gently to mix, then heat for 5- 10 minutes at 65°C to denature the RNA. To each RNA sample add 30μl of master mix. Leave the samples at 25°C for 10 minutes, then incubate at 42°C for 1 hour. The reverse transcriptase reaction is now complete, each microfuge tube should now contain the cDNA complementary to the starting RNA. Denature the cDNA for 30 seconds at 95°C, then place in a CoolBox XT workstation at 4°C or on ice until you are ready for Part 2.

Part 2. PCR

NOTE: This protocol is for standard non-quantitative PCR. For qPCR follow the steps in the qPCR protocol here. At this stage you can also substitute in your own PCR protocol if you are working in different volumes of plate formats (e.g. 5 μL volume 384 well plate).

Unless you are using a DNA polymerase designed for Hot-Start PCR, the work MUST be performed at 4°C or less to reduce nuclease activity and prevent degradation. NOTE: Even when using Hot-Start PCR, if low yield, no amplification or non-specific amplification is observed then this can often be eliminated by adopting a pseudo-hot start protocol, i.e Hot-Start set-up conducted at 4°C.

2.1.         Create a master mix of reagents. This should be carried out at 4°C ideally in an ice-free CoolBox 2XT PCR or on ice with a CoolRack M15 module and CoolRack PCR module. The volumes below are per cDNA sample, e.g., for 10 samples multiply each component by 10, plus a small excess. Once you have added all of the reagents together, vortex gently to mix, and retain at 4°C or on ice while you retrieve or prepare the RNA samples.

2.2.         1.5μL 10x PCR buffer

2.3.        0.5μL forward primer (100 ng)

2.4.        0.5μL reverse primer (100 ng)

2.5.        10.3μL distilled H2O

2.6.        0.2μL Taq Polymerase

2.7 Aliquot 6.0μL of your prepared cDNA into each well of your PCR reaction plate, or tubes.

2.8. Add 13μL reaction mix to each sample. Maintain the samples at 4°C until you are ready to transfer them onto the PCR thermocycler and start the reaction.

2.9 Perform the PCR reaction at the recommended annealing temperature for your primers for the recommended number of cycles.


See other protocols:

BioCision RNA isolation protocol
BioCision qPCR protocol
BioCision General PCR application
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Learn more about:

CoolRack tube modules

CoolBox XT and CoolBox 2XT ice-free cooling workstations

Ice pans and buckets

TruCool ergonomic microfuge tubes

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