RT-PCR

Reverse transcription (RT-PCR): Reverse transcription technology, in simple terms, uses RNA as a template to synthesize DNA. RT-PCR is a modification and addition to the central dogma of molecular biology^[1]. Reverse transcription can be expressed as: reverse transcription and reverse transcription. (1) Reverse transcription is the process of artificially extracting RNA and using it as a template to synthesize DNA in vitro during the research process. (2) Reverse transcription is an autonomous behavior of RNA virus, which uses its own genetic material as a template to synthesize DNA in vivo.

RT-PCR is simple, specific, and sensitive, and can be used to detect gene expression levels and expression differences in cells; detect RNA virus content; clone cDNA sequences of specific genes. At present, real-time reverse transcription polymerase chain reaction has been developed, and fluorescent reporter molecules are added in reverse transcription PCR to monitor the production of amplification products in each cycle of PCR reaction. This method does not require the detection of amplification products by gel electrophoresis, and has simplicity, specificity, sensitivity, and high-throughput adaptability^[2].

MCE has not independently verified the accuracy of these methods. They are for reference only.

Experimental elements: template RNA, primers, reverse transcriptase and others ( dNTP and buffer ) .

Experimental process: mRNA is used as a template for replication, and a DNA strand complementary to mRNA (ie cDNA) is synthesized through the action of reverse transcriptase (I). After the first strand of cDNA is synthesized, the mRNA is degraded, and DNA polymerase (II) continues to use the first strand of cDNA as a template to synthesize the second strand of cDNA, thereby obtaining a double-stranded cDNA molecule.

1. Template RNA

The quality of the template RNA determines the concentration, purity and integrity of the product cDNA . To avoid conditional constraints on the template RNA, the following should be noted:

(1) Obtain high purity and integrity total RNA. Reduce the restriction of salt ions, metal ions, ethanol, phenol and other inorganic impurities on the efficiency of reverse transcriptase. Avoid fragmented, incomplete RNA templates that affect cDNA length and quality. Note: MCE provides spin-column RNA extraction kit (HY-K1082) . The kit can extract viral DNA/RNA, and the obtained products are suitable for PCR, RT-PCR, qPCR, qRT-PCR, etc.

(2) Added incubation treatments. The secondary structure of the RNA can cause the reverse transcriptase to terminate the reaction prematurely, or fall off the template. Adjust temperature conditions, incubate RNA, alter RNA secondary structure, and inhibit secondary structure formation.

(3) Genomic DNA (gDNA) removal. Add DNase I pretreated RNA to remove gDNA. And pay attention to inactivating the endonuclease before reverse transcription to ensure the purity of the product cDNA. Note: MCE provides a reverse transcription kit (HY-K0511A) with premixed DNA digestion enzymes. The kit is equipped with a gDNA digester, which can eliminate genomic DNA contamination and ensure the uniqueness of cDNA in the product.

2. Primer

There are three types of primers for the reverse transcription process^[3]:

(1) Poly-thymidine primers (Oligo (dT)): It can synthesize full-length cDNA. The template RNA is required to have high integrity, poly(A) tail and simple secondary structure. It can be applied to construct eukaryotic cDNA library and clone full-length cDNA.

(2) Random Primers: High PCR yield and wide applicability. Suitable for prokaryotic RNA, rRNA, tRNA, circRNA, microRNA, degraded RNA, and RNA with high GC content and complex secondary structure. However, the specificity is poor and the synthetic DNA fragments are short. It can be applied to the reverse transcription of circRNA, or used in conjunction with Oligo (dT) .

(3) Gene-specific primers (GSP): strong specificity, only amplifies the target fragment; requires a higher annealing temperature. Can be used in one-step RT-PCR reactions.

3. Reverse transcriptase

The discovery of reverse transcriptase complemented the central dogma and won the 1975 Nobel Prize in Physiology and Medicine . Currently, there are two main types of reverse transcriptase: one is AMV reverse transcriptase derived from avian myeloblastosis virus (AMV), and the other is MoMLV reverse transcriptase derived from Moloney murine leukemia virus (MoMLV).

1. RT-PCR usually has a one-step or two-step method, see Table 2 for details.

2. Taking the RT Master Mix for qPCR II (gDNA digester plus) (HY-K0511A) kit as an example, the reaction procedure is shown in Table 3. The example kit comes with 4× Super RT Mix, which contains gDNA digester inhibitor.

NOTE: The recommended reverse transcription temperature is 55ºC. For templates with high GC content or complex structures, the reverse transcription temperature can be increased to 60ºC.

1. Enzyme requirements

(1) gDNA digester Mix or Super RT Mix contain high concentrations of glycerol. Before use, it should be collected by short centrifugation to the bottom of the reaction tube, and gently pipetted with a pipette to mix well, and then pipet accurately.

(2) All reaction systems should be prepared on ice.

2. Experimental conditions

(1) It is recommended to use RNase-free tips and centrifuge tubes, special utensils and sterile water to avoid RNase contamination;

(2) The product can be used immediately for qPCR reaction, or stored at -20°C, and used within half a year; for long-term storage, it is recommended to store at -70°C after aliquoting. cDNA should avoid repeated freezing and thawing.

(1) Determination of RNA purity and concentration;

(2) qRT-PCR assay and analysis.