Reverse transcription-polymerase chain reaction

Summary

PCR refers to the process of replicating a daughter strand of DNA complementary to the template DNA sequence in vitro through a three-step cycle of denaturation, annealing and extension catalyzed by DNA polymerase, using the parental DNA as a template and a specific primer as the extension starting point, and it can amplify any target DNA fragments in vitro in a rapid and specific manner. Qualitative, semi-quantitative and quantitative PCR techniques are widely used in biology and medicine. At present, semi-quantitative PCR methods mainly include RT-PCR.

Principle

The basic principle of RT-PCR in semi-quantitative PCR is to synthesize a cDNA strand using RNA as a template and reverse transcriptase, and then use the cDNA as a template to synthesize the target DNA fragments by PCR amplification.

Operation method

Reverse transcription-polymerase chain reaction

Materials and Instruments

Reagents:
Template RNA, reverse transcriptase and primers
Instrument:
PCR instrument

Move

The basic process of RT-PCR can be divided into the following steps:

(i) Total RNA extraction from animal tissues/cells (TRizol method):

A. Take 0.1-0.2 g of fresh animal tissue and place it in a mortar.

A. Take 0.1~0.2 g of fresh animal tissue in a mortar, cut the tissue with scissors, add a small amount of liquid nitrogen into the mortar, grind it rapidly, wait until the tissue becomes soft, then add a small amount of liquid nitrogen, grind it again, and then make it into powder, add 1 ml of TRizol to every 100 mg of tissue, and then homogenize the tissue rapidly for 15~30s in an ice bath, in order to sufficiently grind the tissue.

The

cell suspension was then pipetted into another microcentrifuge tube and allowed to stand for 5 min at room temperature.

B

If the cells are adherent, incubate for a predetermined period of time and then discard the culture medium completely. Add TRizol reagent directly onto the adherent cells and leave at room temperature for 10 min. For suspension culture, collect the cells by centrifugation and resuspend and lysed with TRizol directly

.

C

Repeatedly blow the lysed tissues/cells, and the lysate is transferred to a new tube and left at room temperature for 5

min, 4°C, 12,000°F, 12,000°C, 12,000°F, 12

^,

000°C, 12,000°C, 12,000°C, 12,000°C, 12,000°C, 12,000°C, 12,000°C, 12,000°C, 12,000°C, 12,000°C, 12,000°F, 12,000°C. The lysate was transferred into a new tube and allowed to stand for 5

min

at room temperature.

D

Transfer the supernatant into a new tube, add 200 μL of chloroform in the ratio of TRizol: chloroform equal to 5:1, mix well by forceful inversion, and allow to stand for 10 min, then allow to stratify, and centrifuge for 15 min at 4°C, 12000 g.

E

Transfer the aqueous phase to a new tube with care. 10 min at room temperature, centrifuge at 12000 g for 10 min at 4℃, and carefully discard the supernatant.

F Add 75% ethanol to the precipitate, shake for a while, and centrifuge at 7500 g for 5 min at 4℃, and carefully discard the supernatant. Let the RNA precipitate dry at room temperature for 5-15 min, then dissolve it in DEPC water and store it in a refrigerator at -70℃.

(ii) RT-PCR reaction:

A

. Reverse transcription

In a microcentrifuge tube, add 2 µg of template RNA, 5 µl of Oligo(dT ₎₁₈ (0.1 µg/ µl), 2 µl of 10 mmol/L dNTP, and 14 µl of DEPC water, and then place the tube on ice for 5 min at 65°C. Add the following to the tube in the following order:
5 *M-MLV Reaction Buffer 4 µl.
Nuclease Inhibitor (25U/µl) 1µl
M-MLV Reverse Transcriptase (100U/µl ) 1µl
Incubate at 37°C for 60 min.

B. PCR Reaction Reverse Transcription Product (Template) 2µl
10X PCR Reaction Buffer 5µl
10 mmol/L dNTP 4µl
Primer (50µmol/L) 1µl
Taq Enzyme (5U/µl ) 0.5µl DEPC Water 37.5µl l
DEPC water 37.5µl
Amplification program: 94℃ 5 min hot start; 94℃ 45s,57℃ 30s,72℃ 1 min, 30 cycles; 72℃ 10 min.

Caveat

PrecautionsPrecautions during RT-PCR experiments are as follows:The RNA obtained by extraction is analyzed for integrity by agarose gel formic acid denaturing electrophoresis.The success or failure of the RT-PCR experiment depends largely on the purity and integrity of the RNA. When extracting RNA from animal tissues/cells, the RNA is separated from other cellular components by treatment with high concentrations of protein denaturants and organic solvents such as phenol and chloroform, and centrifugation. During the extraction process, endogenous and exogenous RNAase activities are inhibited to protect the RNA molecule from degradation.PCR-amplified DNA fragments can be resolved and detected by agarose gel electrophoresis, and can be analyzed by grayscale analysis using Total Lab software, which expresses the relative level of mRNA in terms of the ratio of the grayscale of the target gene to that of the internal reference gene.The key step in RT-PCR is the reverse transcription of the RNA, followed by the partial degradation of the RNA portion of the DNA-RNA heterodimer catalyzed by ribonuclease H (RNaseH). Uncleaned proteins can bind to the RNA and interfere with the reverse transcription and PCR reactions. If trace amounts of DNA are contaminated in the RNA template, amplification results in a PCR product with nonspecific DNA.Common Problems and SolutionsRNA molecules are susceptible to degradation: Extractions must be performed in an RNase-free environment, and RNA extraction reagents should be prepared with diethypyrncarbonate (DEPC)-treated water to remove residual RNAase.

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