RealtimePCR

實驗概要

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

實驗原理

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.

主要試劑

SYBR [ Details：from Sigma S4438]

主要設備

ABI Prism SDS 7000

實驗材料

cDNA

實驗步驟

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.

注意事項

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.