实验概要
The exponential amplification via reverse transcription polymerase chain reaction provides for a highly sensitive technique in which a very low copy number of RNA molecules can be detected. RT-PCR is widely used in the diagnosis of genetic diseases and, semiquantitatively, in the determination of the abundance of specific different RNA molecules within a cell or tissue as a measure of gene expression. Northern blot analysis is used to study the RNA's gene expression further. RT-PCR can also be very useful in the insertion of eukaryotic genes into prokaryotes. Because most eukaryotic genes contain introns which are present in the genome but not in the mature mRNA, the cDNA generated from a RT-PCR reaction is the exact (without regard to the error prone nature of reverse transcriptases) DNA sequence which would be directly translated into protein after transcription. When these genes are expressed in prokaryotic cells for the sake of protein production or purification, the RNA produced directly from transcription need not undergo splicing as the transcript contains only exons. (Prokaryotes, such as E. coli, lack the mRNA splicing mechanism of eukaryotes).
RT-PCR is commonly used in studying the genomes of viruses whose genomes are composed of RNA, such as Influenzavirus A and retroviruses like HIV
实验原理
Reverse transcription polymerase chain reaction (RT-PCR) is a variant of polymerase chain reaction (PCR). It is a laboratory technique commonly used in molecular biology where a RNA strand is reverse transcribed into its DNA complement (complementary DNA, or cDNA) using the enzyme reverse transcriptase, and the resulting cDNA is amplified using PCR.
主要试剂
SYBR [ Details:from Sigma S4438]
主要设备
ABI Prism SDS 7000
实验材料
cDNA
实验步骤
1. Set up the experiment and the following PCR program on ABI Prism SDS 7000. Do not click on the dissociation protocol if you want to check the PCR result by agarose gel.
1)94°C 2 min, 1 cycle
2)94 °C 15 s -> 55 °C 30 s -> 72 °C 30 s, 40 cycles
3)72°C 10 min, 1 cycle
2. A real-time PCR reaction mixture: 50 ml. (in our lab, to save reagent, use 25 ml)
1)12.5 ml SYBR Green Mix (2x)
2)1 ml cDNA
3)1 ml primer pair mix
4)10.5 ml H2O
3. After PCR is finished, remove the tubes from the machine. The PCR specificity is examined by 3-4% agarose gel.
4. Put the tubes back in SDS 7000 and perform dissociation curve analysis.
注意事项
Several factors should be considered when designing Real time primers:
1. The primers should flank but not overlap the probe sequence.
2. Optimal primer length ranges from 15-30 bases.
3. Avoid complementary internal sequences to minimize formation of secondary structure.
4. Targets an amplicon length of 75 to 150 bp
5. 50 to 60% GC content
6. Melting Temperature (Tm) above 50C
7. Verify specificity
The primer used in normal PCR maybe not fit Real time PCR.So first check the primer use normal PCR, then check on gel, then try them on Real time PCR to make sure there is no primer dimer.
Template In normal PCR, check the template useing housekeeping gene. The good cDNA should have no genomic DNA contamination.