Real Time PCR and its Application in Plant Pathology-II

  Protocol for Real-Time PCR

RT-PCR can be carried out by the one step RT-PCR protocol or the two step RT-PCR protocol. Use only intact, high quality RNA for the best results.

A. One step RT-PCR protocol: one step RT-PCR protocol take mRNA targets (up to 6 kb) and subjects them to reverse transcription and then PCR amplification in a single test tube.

1. Select a one step RT-PCR kit, which should include a mix with the reverse transcriptase and the PCR system such as Taq DNA Polymerase and a proofreading polymerase
2. Obtain all necessary materials, equipment and instruments
3. Prepare a reaction mix, which will include dNTPs, primers, template RNA, necessary enzymes and a buffer solution
4. Add the mix to a PCR tube for each reaction. Then add the template RNA
5. Place PCR tubes in the thermal cycler to begin cycling. The first cycle is reverse transcription to synthesize cDNA. The second cycle is initial denaturation. During this cycle reverse transcriptase is inactivated. The next 40-50 cycles are the amplification program, which consists of three steps: denaturation, annealing and elongation
6. The RT-PCR products can then be analyzed with gel electrophoresis

B. Two step RT-PCR protocol: two steps RT-PCR, as the name implies, occurs in two steps. First the reverse transcription and then the PCR. This method is more sensitive than the one step method. 

Step 1:

1. Combine template RNA, primer, dNTP mix, and nuclease free water in a PCR tube
2. Add RNase inhibitor and reverse transcriptase to the PCR tube
3. Place PCR tube in thermal cycler for one cycle and then inactivating reverse transcriptase
4. Proceed directly to PCR or store on ice until PCR can be performed

Step 2:

1. Add a master mix (containing buffer, dNTP mix, MgCl_2, Taq polymerase and nuclease free water) to each PCR tube
2. Add appropriate primer
3. Place PCR tubes in thermal cycler for 30 cycles of the amplification program
4. The RT-PCR products can then be analyzed with gel electrophoresis

                                                                              Continued........