Application of mRNA reverse transcription amplification of cDNA (RT-PCR)

Summary

RT-PCR can be used to: (1) construct large-capacity cDNA libraries; (2) identify mutations and polymorphisms in transcribed sequences; (3) measure the intensity of gene expression.

Operation method

Application of mRNA reverse transcription amplification of cDNA (RT-PCR)

Principle

Enzyme-catalyzed reverse transcription of RNA into the first strand of cDNA. An oligodeoxynucleotide primer hybridizes to the mRNA and is then catalyzed by RNA-dependent DNA polymerase to synthesize a complementary copy of the cDNA, which can be further used for PCR amplification. Depending on the purpose of the experiment, primers synthesized for the first strand of single-stranded cDNA can be designed to hybridize with specific target genes, or random primers can be used to hybridize with all mRNAs.

Materials and Instruments

Tissue Cells
Amplification buffer Chloroform Storage solution for dNTP Ethanol Phenol Chloroform Placenta RNase inhibitor Reverse transcriptase DNA polymerase Exogenous reference RNA
Agarose gel or polyacrylamide gel Shielded tip Tubes or microtitre plates Positive displacement pipettes PCR instruments Water baths

Move

I. Total RNA extraction

See "extraction of total RNA".

Synthesis of cDNA first strand

At present, there are many kinds of cDNA first strand reagent kits for sale, the principle of which is basically the same, but the operation steps are different. Now take the SuperScriptTM Preamplification System for First Strand cDNA Synthesis kit provided by GIBICOL as an example.

1. In a 0.5 ml microcentrifuge tube, add 1-5 μg of total RNA, add appropriate amount of DEPC H2O to make the total volume of 11 μl. Add 10 μM Oligo (dT) 12-18 1 μl to the tube, mix gently and centrifuge. 2;

2. Heat at 70°C for 10 min and immediately insert the microcentrifuge tube into an ice bath for at least 1 min;

3. Take 0.5 ml PCR tube, add the following reagents sequentially: first strand cDNA 2 μl; upstream primer (10 pM) 2 μl; downstream primer (10 pM) 2 μl; dNTP (2 mM) 4 μl; 10 × PCR buffer 5 μl; Taq enzyme (2 u/μl) 1 μl. mix gently and centrifuge. incubate at 42℃ for 2-5 min;

4. Add 1 μl of Superscript II and incubate at 42℃ for 50 min;

5. Heat at 70°C for 15 min to terminate the reaction;

6. Insert the tube into ice, add RNase H 1 μl, incubate at 37 ℃ for 20 min, and degrade the residual RNA. store at -20℃ for spare use.

PCR

1. Take 0.5 ml PCR tube and add the following reagents: first strand cDNA 2 μl; upstream primer (10 pM) 2 μl; downstream primer (10 pM) 2 μl; dNTP (2 mM) 4 μl; 10 × PCR buffer 5 μl; Taq enzyme (2 u/μl) 1 μl. 2. Add the appropriate amount of ddTCP (2 pM), then add RNase H 1 μl;

2. Add appropriate amount of ddH2O to make a total volume of 50 μl. Mix gently and centrifuge;

3. Set up the PCR program. Amplify 28-32 cycles under appropriate temperature parameters. In order to ensure the reliability and accuracy of the experimental results, a pair of primers specific for the internal reference (e.g., G3PD) can be added to PCR amplification of the target gene, and the internal reference DNA can be amplified at the same time as a control;

4. Electrophoresis identification: perform agarose gel electrophoresis and observe the results under UV light;

5. Density scanning and result analysis: use gel image analysis system to scan the density of electrophoretic bands.

Caveat

1. In the course of the experiment, the degradation of RNA should be prevented and the integrity of RNA should be maintained. During the extraction of total RNA, care should be taken to avoid the breakage of mRNA;2. In order to prevent non-specific amplification, a negative control must be set;3. The setting of internal reference: mainly for the quantification of target RNA. Commonly used internal parameters are G3PD (glyceraldehyde-3-phosphate dehydrogenase), β-Actin (β-actin) and so on. The purpose is to avoid RNA quantification errors, spiking errors and errors caused by uneven amplification efficiency in each PCR reaction system and temperature differences between wells;4. PCR cannot enter the plateau period, and the plateau effect is related to the length, sequence, secondary structure of the amplified target gene and the number of target DNA starts. Therefore, the number of cycles of platform effect for each target sequence should be determined by separate experiments;5. Prevent DNA contamination: use DNA enzymes to treat RNA; where possible, place PCR primers in different exons of the gene to eliminate gene and mRNA covariance.

Common Problems

1. Selection of reverse transcriptase

(1) Money murine leukemia virus (MMLV) reverse transcriptase: strong polymerase activity and relatively weak RNAase H activity.

The

optimal temperature of action is 37℃;

(2) Avian myeloblastoma virus (AMV) reverse transcriptase: has strong polymerase activity and RNAase H activity.

The

optimal temperature of action is 42°C;

(3) Heat-stable reverse transcriptase of thermophilic microorganisms such as Thermus thermophilus, Thermus flavus, etc.: in the presence of Mn2+, it allows high-temperature reverse transcription of RNA in order to eliminate the secondary structure of the RNA template;

(4) RNase H-mutants of MMLV reverse transcriptase: the trade names are Superscript and SuperScript II. This enzyme is able to convert a larger portion of RNA into cDNA than the other enzymes, a feature that allows the synthesis of longer cDNAs from mRNA templates containing secondary structures that are difficult to reverse transcribe at low temperatures.

2

. Selection of primers for synthesizing cDNAs

(1) Random hexameric primers: When it is difficult to copy the full-length sequence of a given mRNA because it contains sequences that cause termination by the reverse transcriptase enzyme, random hexameric primers, a non-specific primer, can be used to synthesize the full-length sequence. Random Hexamer Primer: When it is difficult to copy the full-length sequence of a specific mRNA because it contains sequences that terminate reverse transcriptase, a random hexamer primer can be used as a non-specific primer to copy the full-length mRNA. Usually 96% of the cDNA synthesized with this primer is derived from rRNA.

(2) Oligo(dT): is a method specific for mRNA. Since most eukaryotic mRNAs have a Poly(A+) tail at the 3' end, this primer pairs with it and only the mRNA can be transcribed. Because Poly(A+) RNA only accounts for 1-4% of the total RNA, the cDNA synthesized by this primer is smaller than that obtained by using random hexamers as primers and in terms of quantity and complexity.

(3) Specific primers: the most specific method of initiation is to use oligonucleotides containing the complementary sequence of the target RNA as primers, if the PCR reaction with two specific primers, the synthesis of the first strand can be initiated by the closest pair of primers to the 3' end of the mRNA. Using such primers produces only the desired cDNA, leading to more specific PCR amplification.

Source "Guide to Molecular Cloning Experiments (Third Edition)" translated by Huang Peitang et al.

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