What is RT-PCR?
What is RT-PCR?
RT-PCR, reverse transcription-polymerase chain reaction (RT-PCR) is a technique that combines reverse transcription of RNA and polymerase chain amplification (PCR) of cDNA.
First, cDNA is synthesized from RNA by the action of reverse transcriptase, and then cDNA is used as a template to amplify and synthesize the target fragment. RT-PCR technology is sensitive and versatile, and can be used to detect the level of gene expression in cells, the content of RNA viruses in cells and directly clone the cDNA sequence of specific genes. The RNA used as template can be total RNA, mRNA, or an in vitro transcribed RNA product. Regardless of the RNA used, it is critical to ensure that the RNA is free of RNase and genomic DNA contamination.
Total RNA from tissues or cells was extracted, and mRNA was used as a template to reverse transcribed into cDNA using Oligo (dT) or random primers and reverse transcriptase. Then use cDNA as a template for PCR amplification to obtain the target gene or detect gene expression.
The process of RT-PCR
RT-PCR can be performed in a one-step or two-step format. In two-step RT-PCR, each step is performed under optimal conditions. cDNA synthesis was first performed in reverse transcription buffer, and then 1/10 of the reaction product was removed for PCR. In one-step RT-PCR, reverse transcription and PCR are performed sequentially under conditions optimized for both reverse transcription and PCR.
1. Selection of reverse transcriptase
① Money Murine Leukemia Virus Reverse Transcriptase
It has strong polymerase activity and relatively weak RNase H activity. The optimum temperature is 37°.
② AMV reverse transcriptase
It has strong polymerase activity and RNase H activity. The optimum temperature is 42°.
2. Selection of synthetic cDNA primers
The primers used for reverse transcription can be selected from random primers, Oligo dT and gene-specific primers according to the specific conditions of the experiment. For short eukaryotic mRNAs that do not have hairpin structures, all three are acceptable. Choice of RT-PCR primers:
① Random primers are suitable for long or hairpin RNAs. Suitable for reverse transcription reactions of all RNAs such as rRNA, mRNA, tRNA, etc. Mainly used for RT-PCR reactions with a single template.
② Oligo dT is available for RNAs with PolyA tails. (Prokaryotic RNA, eukaryotic rRNA, and tRNA do not have PolyA tails.) Since Oligo dT is bound to PolyA tails, the quality of RNA samples is high, and even a small amount of degradation will result in full-length cDNA synthesis The amount is greatly reduced.
③ Gene-specific primers are primers that are complementary to the template sequence and are suitable when the target sequence is known.
What are the influencing factors of RT-PCR?
The RT-PCR reaction is affected by many factors, such as the concentration of magnesium sulfate, the temperature of primer annealing, the number of cycles of amplification, etc.
1. It is recommended to select 0.5-3.0 mM (0.5 mM difference) magnesium sulfate for preliminary experiments.
2. For primers with higher Tm, it is beneficial to increase the temperature during annealing and extension. Higher temperatures are beneficial to reduce non-specific primer binding, thereby increasing the yield of specific products.
3. Most target RNAs can be observed after 40 rounds of PCR reactions. But if the target RNA is too rare, or there is only very little starting material, it may be necessary to increase the number of amplifications to 45-50 times.
RT-PCR system supplier:
Geneture medical specialized in the field of nucleic acid extraction and PCR detection system, main products involved in：
1.Nucleic Acid Extraction Kit (Magnetic beads method)
2.Nucleic Acid Extractor （32T, 96T）
3.PCR fluorescence quantitative analyzer (16T, 96T)
4.PCR detection reagent
5.SARS-CoV-2 antigen rapid test kit
6.Lab consumables: deep well plate, mag-rod sleeve, pipette tips, VTM, swabs, PCR tubes, PCR plate, centrifuge tubes, cryogenic Vials, saliva collection and so on.
Please don’t hesitate to contact with us to get a quotation.