Quantitative qPCR (quantitative polymerase chain reaction) detection method and kit for tRNA (transfer ribonucleic acid) aminoacylation level

A detection method and kit technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of limiting the application of tRNA detection methods, strong dependence on test equipment, complex structure, etc., and achieve remarkable results Technical advantages and application prospects, simplification of primer set design, effect of expanding species range

Pending Publication Date: 2022-08-02
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0005] Third, redundant tRNA gene copies are encoded in human, chicken, and duck nuclear DNA, and high-throughput detection of all tRNAs is very complicated;
[0007] Fifth, tRNA molecules are short and complex in structure, and the steps of obtaining cDNA by conventional methods are lengthy, which greatly limits the application of tRNA detection methods
[0008] Sixth, the tRNA that actually participates in the translation function in the cell is the aminoacylated tRNA, but there are both aminoacylated tRNA and non-aminoacylated tRNA in the cell, and conventional methods cannot specifically detect the aminoacylation level of tRNA
[0009] At present, although high-throughput sequencing detection methods for tRNA detection and tRNA aminoacylation have been established, the test equipment is highly dependent, the test cycle is long, and the price is expensive, which greatly limits the research on the biological function of tRNA.
At present, there is no report on the detection of aminoacylation level using conventional qPCR technology

Method used

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  • Quantitative qPCR (quantitative polymerase chain reaction) detection method and kit for tRNA (transfer ribonucleic acid) aminoacylation level
  • Quantitative qPCR (quantitative polymerase chain reaction) detection method and kit for tRNA (transfer ribonucleic acid) aminoacylation level
  • Quantitative qPCR (quantitative polymerase chain reaction) detection method and kit for tRNA (transfer ribonucleic acid) aminoacylation level

Examples

Experimental program
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Effect test

Embodiment 1

[0082] Detection of tRNA-Cys-GCA and tRNA-Ser-GCU aminoacylation levels after duck hepatitis virus (DHAV) infection by qPCR

[0083] 1. Test material:

[0084] Chicken embryo fibroblasts were prepared according to conventional methods, RNA extraction: EZ-10 DNAaway RNA mini-extraction kit, deacylation buffer (50ml): 5×Tris-HCl (100mM, pH9.0), annealing buffer (50ml) ): 10×Tris-HCl (50mM, PH8.0), T4 RNA ligase (dsRNA ligase) (NEB, M0239L), 5×Prime Script RT MasterMix (Takara, RR036Q), Taq Pro Universal SYBR qPCR Master Mix ( Vazyme: Q712).

[0085] 2. Test method (refer to figure 1 the second half)

[0086] 2.1 The total RNA of chicken embryo fibroblasts DHAV-infected cells was extracted using the kit.

[0087] Take 4 μl RNA (concentration of 214ng / μl), add 1 μl 5× deacylation buffer and incubate at 37°C for 40 minutes, then add 10 μl TE buffer to elute to obtain total RNA containing deacylated tRNA (7 μl contains 400ng RNA) The untreated RNA contains both unaminoacylated...

Embodiment 2

[0106] Dynamic changes of nuclear and mitochondrial-encoded tRNA groups after duck hepatitis virus (DHAV) infection by qPCR

[0107] 1. Test material

[0108] Chicken embryo fibroblasts were prepared according to conventional methods, RNA extraction: EZ-10 DNAaway RNA mini-extraction kit, deacylation buffer (50ml): 5×Tris-HCl (100mM, pH9.0), annealing buffer (50ml) ): 10×Tris-HCl (50mM, PH8.0), T4 RNA ligase (dsRNA ligase) (NEB, M0239L), 5×Prime Script RT MasterMix (Takara, RR036Q), Taq Pro Universal SYBR qPCR Master Mix ( Vazyme: Q712).

[0109] 2. Test method (refer to figure 1 )

[0110] 2.1 Using the kit to extract DHAV-infected cells and non-infected cells of chicken embryo fibroblasts.

[0111] The specific steps are the same as the kit instructions. The present invention combines the amino acid removal step and RNA extraction to simplify the steps, that is, add 10 μl of deacylation buffer before elution and incubate at 37°C for 40 minutes, and then add 20 μl of TE ...

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Abstract

The invention relates to a quantitative qPCR (quantitative polymerase chain reaction) detection method and kit for the aminoacylation level of tRNA (transfer ribonucleic acid), which comprises the following steps: (1) extracting total RNA (ribonucleic acid) without removing amino acid from the tRNA; (2) removing amino acid from tRNA in the total RNA to obtain total RNA containing all deacylated tRNA; according to the present invention, the amino acid sequence and the amino acid sequence are respectively connected with the same tRNA linker, qPCR detection is performed after reverse transcription, the difference value of the Ct value indicates the amino acylation level of the tRNA, and the amino acid load level of the tRNA can be calculated through internal reference gene correction and data standardization processing. Besides, the invention innovatively provides a simple co-reverse transcription method of all tRNA and mRNA for transcribing cell nucleus and mitochondria, the technical barrier of tRNA aminoacylation detection is overcome, and quantitative detection of the tRNA aminoacylation level can be realized by using a conventional qPCR technology.

Description

technical field [0001] The invention belongs to the technical field of molecular biochemistry, and in particular relates to a quantitative qPCR detection method and kit for tRNA aminoacylation level. Background technique [0002] tRNA is the carrier of amino acids, mainly involved in the recognition of the genetic code, and is an important molecule linking mRNA and protein expression. At present, 415, 271, and 361 tRNA genes are known to be encoded in the nuclear genomes of human, chicken, and duck cells, respectively; however, the mitochondrial genomes all encode 22 kinds of tRNA. Due to the small fragment of tRNA molecules, about 73-96nt, conventional methods cannot detect such small RNA molecules. In addition, tRNA participates in mRNA translation in cells and actually functions in the form of aminoacylated tRNA. At present, tRNA mainly has the following characteristics, which make the detection of tRNA and its aminoacylation level difficult: [0003] First, the 2' hydr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11C12Q1/6851
CPCC12Q1/6888C12Q1/6851C12Q2600/158C12Q2600/166C12Q2561/113C12Q2531/113C12Q2521/107C12Q2563/107C12Q2525/191
Inventor 欧旭敏董静文程安春潘秋卫汪铭书彭文婧林晓铭
Owner SICHUAN AGRI UNIV
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