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Overview of RNA extraction technology
RT-PCR is a combination of reverse transcription (RT) of RNA and polymerase chain amplification (PCR) of cDNA, which has been rapidly developed and widely used in recent years. First, a complementary DNA strand (cDNA) was synthesized by reverse transcriptase using RNA as a template, and the target fragment was synthesized by PCR amplification using cDNA as a template. RT-PCR technology is sensitive and versatile. The obtained cDNA can be used for gene amplification, cloning, detection of gene expression levels, and content of intracellular RNA virus.
A. Reverse transcriptase (Reverse Transcriptase, RTase)
A kind of retrovirus with RNA as a genetic material is widely existed in nature. The reverse transcriptase carried by it is a kind of DNA polymerase that uses RNA as a template to synthesize cDNA. At present, Avian Myeloblastosis Virus (AMV) reverse transcriptase (AMV RT) and Moloney Murine L (Leukemin Virus, M) are widely used in life science research. -MLV) Reverse transcriptase (M-MLV RT) and its engineered recombinant M-MLV (RNase H -). AMV RT has the characteristics of high reverse transcription activity and high optimum reaction temperature (41-50 °C), which can better overcome the problem of reverse transcription caused by template secondary structure. However, due to the high RNase H activity of AMV RT The RNA strand in the cDNA-RNA complex is easily degraded, resulting in a short cDNA fragment, generally about 1 kb. The RNase H activity of M-MLV RT is low, and the synthesized cDNA can reach 3~4 kb, which is suitable for the synthesis of full-length cDNA. However, the amplification efficiency of M-MLV RT is low, and the optimum reaction temperature is about 37 °C, so the reverse transcription effect of RNA template with complex secondary structure is not good. The site-directed mutagenesis of M-MLV RT (RNase H-) substantially eliminated the RNase H enzyme activity and increased the optimal reaction temperature of the enzyme to 42 ° C, which greatly improved the elongation and yield of the cDNA strand, and was more suitable. Obtaining the complete gene. The StarScript Reverse Transcriptase (Cat. No. A211) provided by our company is a MNase H enzyme activity-deficient M-MLV RT with multiple site-directed mutagenesis and high amplification ability, which is very suitable for longer cDNA synthesis and high ratio. Construction of a full-length cDNA library, etc.
B. Template RNA
The RNA sample used as a template in the reverse transcription reaction may be extracted total RNA, mRNA or an in vitro transcribed RNA product. There are various methods for extracting RNA, such as a single phase reagent method (TRIGene, Cat. No. P118; TRI Gene LS, Cat. No. P119), a column purification method, a magnetic bead method, and the like. Regardless of which method is used, the most critical factor is inhibition of RNase activity and maximal removal of genomic DNA contamination.
C. Primer
Primers for reverse transcription should be selected depending on the specific circumstances of the experiment. Commonly used primers are Oligo (dT) 12-18 , random primer (Random Hexamer) and Gene-specific primer (GSP). For short eukaryotic mRNAs that do not have a hairpin structure, three primers can be used. Oligo (dT) 12-18 is only applicable to RNAs with PolyA tails, and is not applicable to RNA of prokaryotic organisms without PolyA tails, rRNAs of eukaryotes, tRNAs, and mRNAs of certain types of eukaryotes. Since the Oligo (dT) 12-18 primer needs to bind to the PolyA tail of the mRNA, the quality of the RNA template is very high, and even a small amount of degradation of the template affects the synthesis amount of the full-length cDNA. Random primers are suitable for any type of RNA template, but due to their low specificity, they are mainly used for RT-PCR reactions of a single template. Gene-specific primers are complementary primers designed according to the template sequence and have good specificity, but only for the case where the target sequence is known.
D. Other influencing factors
The reverse transcription reaction is affected by a number of factors, such as Mg 2+ concentration, primer annealing temperature, number of cycles of amplification, and the like. For primers having a higher T m value, while increasing the temperature of the annealing and extension reaction advantageously. Higher temperatures favor the reduction of non-specific primer binding, thereby increasing the yield of specific products. For the optimization of the reverse transcription reaction, see the section "1.7 RT-PCR Common Problems and Solutions".
E. One-step and two-step RT-PCR
RT-PCR can be achieved in the form of a one-step or two-step method. One-step method is to complete reverse transcription and PCR amplification in the same tube, reducing the loading step and helping to reduce pollution. Since all cDNA products are used for PCR amplification, their sensitivity is very high, especially for the detection of large numbers of samples and real-time quantitative PCR. The two-step method separates reverse transcription from PCR amplification and provides greater flexibility in selecting DNA polymerases (such as high fidelity, high specificity, long fragments, etc.) and primers.