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Reverse transcription method, reverse transcription kit and application thereof

A technology of reverse transcription and kits, applied in the biological field, can solve the problems of inconvenient popularization and high overall cost, achieve good results, reasonable and appropriate settings, and reduce the difficulty of operation

Pending Publication Date: 2017-06-20
唐旌生物科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Based on the above factors, the overall cost of the classic method is high, and it has a certain technical threshold, which is not easy to popularize in primary hospitals and small companies

Method used

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  • Reverse transcription method, reverse transcription kit and application thereof
  • Reverse transcription method, reverse transcription kit and application thereof
  • Reverse transcription method, reverse transcription kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] What embodiment 1 adopted was NP-40 lysis solution; What embodiment 2 adopted was NP-40 lysis solution and alkali lysis reagent; What embodiment 3 adopted was alkali lysis reagent; What embodiment 4 adopted was NP-40 lysis solution; What embodiment 5 adopts is alkaline lysis reagent; what embodiment 6 adopts is NP-40 lysate, what embodiment 7 adopts is NP-40 lysate.

[0060] Embodiment 1 Comparison of traditional RNA extraction method and the method of the present invention

[0061] Using 293T cells, using the traditional Trizol cracking and extracting RNA method and the method of the present invention to extract RNA respectively, and then using MMLV reverse transcriptase to reverse transcribe into cDNA, and then quantitatively detect the internal reference genes Gapdh and Tp53 genes by quantitative PCR, and in order to To further verify this result, the above two gene fragments were amplified by common PCR method, and then DNA agarose gel electrophoresis was performed ...

Embodiment 2

[0065] Example 2 Comparison Between Two Kinds of Cell Lysis Reagents

[0066] In order to test whether different lysates can achieve the same effect, although the Trizol lysate used in common RNA extraction can lyse cells to the greatest extent, it will also destroy the protein structure and affect subsequent experiments. Therefore, we continue to study and try new methods. Lysis methods and lysis reagents. After groping, two kinds of cell lysis reagents were found: NP-40 lysis reagent and alkali lysis reagent. Using 293T cells, the alkaline lysate KOH, NP-40 lysate and the RNA extracted by Trizol were compared, and MMLV reverse transcriptase was used for reverse transcription, and the quantitative PCR method was used to quantitatively detect the gene of Tp53, and in order to further verify this As a result, ordinary PCR and DNA agarose gel electrophoresis were used for detection.

[0067] Quantitative PCR test results see image 3 , DNA agarose gel electrophoresis test res...

Embodiment 3

[0068] Example 3 Comparison of MMLV Reverse Transcriptase and AMV Reverse Transcriptase

[0069] Using 293T cells, MMLV reverse transcriptase and AMV reverse transcriptase were used to detect the internal reference gene Gapdh, and the experiments were carried out respectively. The products were detected by quantitative PCR, and DNA agarose gel electrophoresis was used to detect the products after ordinary PCR.

[0070] Quantitative PCR test results see Figure 5 , DNA agarose gel electrophoresis test results see Image 6 . The results showed that the effect was comparable using MMLV reverse transcriptase or AMV reverse transcriptase.

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Abstract

The invention relates to a reverse transcription method, a reverse transcription kit and application thereof. The reverse transcription kit contains NP-40 lysate or an alkaline lysis agent; the lysis agent is the NP-40 lysate; the formula of the NP-40 lysate comprises RNase-Free H2O, NP-40 and RNasin, wherein the mass concentration of the NP-40 is 0.2-5%, and the concentration of the RNasin is 1-10U; the formula of the alkaline lysis agent is prepared from RNase-Free H2O and KOH, wherein the concentration of the KOH is 0.00001-0.01M; the reverse transcription method comprises the steps of firstly, carrying out lysis on a sample by using the lysis agent; then, directly carrying reverse transcription on the sample, subjected to lysis, by using a reverse transcription reagent. The method is a brand-new method for acquiring complementary deoxyribonucleic acid (cDNA) from the sample; after the method is adopted, the operation process is simplified, the operation difficulty is reduced, the experimental time is greatly shortened, and the cost is lowered.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a reverse transcription method, a reverse transcription kit and applications thereof. Background technique [0002] Reverse transcription, also known as reverse transcription, is the process of synthesizing cDNA using RNA as a template through reverse transcriptase. It is a special way of DNA biosynthesis and is catalyzed by reverse transcriptase. American scientists H.M.Temin and D.Baltimore discovered reverse transcriptase in 1970, and won the Nobel Prize in Physiology and Medicine in 1975 for this. The discovery of reverse transcription has greatly promoted the development of molecular biology and genetic engineering technology. It is indispensable in molecular and cell biology experiments such as the construction and expression of eukaryotic or prokaryotic target genes, the construction of cDNA libraries, and the detection of gene sequences. The tools, together with Taq enzymes,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1096
Inventor 沈君炜陈光风史秀娟胡婧
Owner 唐旌生物科技(上海)有限公司
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