RT-PCR is a technique that combines the reverse transcription of RNA with the PCR of cDNA. This article introduces the related experimental steps of RT-PCR, hoping to help your academic research.

1.The reaction fluid is modulated in the PCR reaction tube according to the following composition.

Reagent	Quantity, for 50µl of reaction mixture
10×One Step RNA PCR Buffer	5µl
25 mM MgCl2	10µl
10 mM dNTP mix	5µl
RNase Inhibitor (40 U/µ l)	1µl
AMV-Optimized Taq	1µl
AMV RTase XL (5 U/µl)	1µl
Upstream specific primer	1µl
Downstream specific primer	1µl
Experimental sample RNA（≤1 µg Total RNA）	1µl
RNase Free dH2O	24µl
RNase Free dH2O	24µl

①. The total volume of the reaction is regulated according to the actual situation, and 20-50 μ l can be made to save reagents;

②.Add the components in the above table to 0.2ml or 0.5ml sterilized PCR thin-walled tube;

③. If the heating cover of PCR instrument is not used, add 30-50 μ l mineral oil to the upper layer of reaction mixture to prevent the sample was evaporated during PCR;

2.React according to the following conditions.

Step	Temperature, °C	Time, min	Number of cycles	Note
Reverse transcription	50	30	1
Reverse transcriptase inactivation	94	2	1
Denaturation	94	0.5	25-35
Annealing	37-65	0.5		The annealing temperature is about 5 °C lower than the theoretical annealing temperature, and then optimized according to the reaction results
Extend	72	Depending on the size of the amplification product		Taq enzyme extends 1000 BP per minute
Final extension	72	10	1

At the end of the reaction, 5 μ l of the amplified sample was taken, and the amplification results were analyzed by agarose gel electrophoresis. The size of the amplified fragment was determined by DNA marker or it was cryopreserved for later analysis.