The Reverse-Transcriptase-Polymerase Chain Reaction (RT-PCR) technique has been widely used in life science research to detect and quantify the specific mRNA from biological samples. One major advantage of RT-PCR is its high sensitivity comparing with other traditional RNA detection and quantification techniques, therefore it is best suitable for detection and quantification of low abundance mRNA. For example, using nested RT-PCR it is possible to amplify and detect mRNA expression from a signle cell. Many applications and techniques, including PCR-ELISA, real-time RT-PCR, laser capture microdissection (LCM), and many other RT-PCR variations are originated from RT-PCR technique. It is an extreamly valuable assets in biomedicine research with wide applications in cancer biology, immunology, and pathogenes identifications.
Basic steps for RT-PCR:
1. RNA isolation:
RNA can be extracted and purified by tissue specimens homogenization followed by guanidine thiocyanate lysis, phenol extraction and alcohol precipitation. Many kits offer alternative to phenol extraction by using spun-column chromatography or magnetic bead for fast and environmental friendly RNA isolation. Most RNA extraction methods however can not gurantee 100% removal of contaminated genomic DNA, which may result in interferences in downstream applications. To eliminate genomic DNA contamination, one can use cesium chloride density gradients ultracentrifugation for RNA purification. Alternatively, most applications use PCR primers that span intro-exon conjunctions for RT-PCR amplification, so that PCR products from mRNA (spliced product without introns) and the genomic DNA (with introns) are readily distinguashible by size.
2. Reverse transcription step:
Reverse transcription can be carried out by reverse transcriptase in the presence of mRNA template and primers. Several types of primers, including oligo (dT) primers, random hexamer primers, and gene specific primers, can be used to synthesize the cDNA.
PCR primer design is pivotal to sucessful RT-PCR. There are several principles regarding PCR primer design:
- It is necessary to minimize the self-complementarity between or within primers to avoid primer secondary structures.
- The annealing temperature for 5' and 3' primers (or upstream/downstream primers, sense/antisense primers) should be closly matched.
- GC contents should be around 50%.
- Primer sequences should be filtered through BLAST search to avoid amplification of other non-specific sequences.
Common PCR reaction condition (in 50 ul reaction):
Sense primer 0.1 uM
Antisense primer 0.1 uM
dNTPs 50 uM
MgCl2 1.5 mM
Taq DNA polymerase 1 Unit
PCR cycle programming:
Denaturation step: 96C for 15 seconds.
Annealing step: 55C for 30 seconds.
Extension step: 72C for 3 min.
Hot start step (optional): 80C for 1 min (before adding Taq DNA polymerase).
For specific applications such as DNA cloning, GC rich region amplification, long PCR etc, one needs to use high fedility DNA polymerase and optmized PCR conditions.
In case of PCR contamination:
It is necessary to use a complete set of freshly prepared PCR reagents (using aerosol-resistant pipet tips) and move the PCR setup area to another region in the lab. Try decontaminating bench surface using bleach or commercially avialble PCR decontamination solution. The use of uracil-N-glycosylase (UNG) in PCR reaction can also decrease PCR contamination risks. Exposing the PCR reaction tube under UV light (254nm) can also help to destroy any DNA contamination in the inner surface of the PCR tube.
- Molecular Biology