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Method for quantitatively expressing gene of porcine single oocyte

An oocyte and gene technology, applied in the field of cell biology, can solve the problems of increased sample pollution, many operation steps, and limited application, and achieve the effect of improving sensitivity, reducing pollution, and good effect

Inactive Publication Date: 2019-01-01
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional qPCR operation has many steps and takes a long time, which increases the possibility of sample contamination during the operation, and it requires a large number of samples, and a quantitative PCR requires dozens of samples, which limits the use of this technology. Application in Precious Samples

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] A method for quantitatively expressing a single pig oocyte gene, comprising the following steps:

[0028] S1: Preparation of Assay Pool (primer pool)

[0029] OCT4, NANOG, SOX2, TEL1, TEL2, TEL3, WDR5, EF1α1, ID2, STELLA, CARML, INO80, P21, P27, P53, P57, GAPDH, H2A, METTL3, METTL14, WTAP, ALKBH5, LIN28, L-MYC The amplification primers of 25 genes including CDX2 and CDX2 were mixed in proportion to make a primer pool, and the respective final concentrations of the amplification primers of the 25 genes were 0.1 μM.

[0030] S2: Configuration of RT-PreAmp Master Mix (Reverse Transcription Reaction System)

[0031] In 4 nucleic acid-free centrifuge tubes, configure 5.0 μl of RT-PreAmp Master Mix (reverse transcription reaction system), and place it on ice for use, then take porcine oocytes with good shape, buffer with DPBS Duchenne's phosphate Wash three times with liquid solution, add one oocyte to each of the four centrifuge tubes, immediately place it in a -80°C refri...

Embodiment 2

[0042]A method for quantitatively expressing a single pig oocyte gene, comprising the following steps:

[0043] S1: Preparation of Assay Pool (primer pool)

[0044] OCT4, NANOG, SOX2, TEL1, TEL2, TEL3, WDR5, EF1α1, ID2, STELLA, CARML, INO80, P21, P27, P53, P57, GAPDH, H2A, METTL3, METTL14, WTAP, ALKBH5, LIN28, L-MYC The amplification primers of 25 genes including CDX2 and CDX2 were mixed in proportion to make a primer pool, and the respective final concentrations of the amplification primers of the 25 genes were 0.1 μM.

[0045] S2: Configuration of RT-PreAmp Master Mix (Reverse Transcription Reaction System)

[0046] In 4 nucleic acid-free centrifuge tubes, configure 5.0 μl of RT-PreAmp Master Mix (reverse transcription reaction system), and place it on ice for use, then take porcine oocytes with good shape, buffer with DPBS Duchenne's phosphate Wash three times with liquid solution, add one oocyte to each of the four centrifuge tubes, immediately place it in a -80°C refrig...

Embodiment 3

[0057] A method for quantitatively expressing a single pig oocyte gene, comprising the following steps:

[0058] S1: Preparation of Assay Pool (primer pool)

[0059] OCT4, NANOG, SOX2, TEL1, TEL2, TEL3, WDR5, EF1α1, ID2, STELLA, CARML, INO80, P21, P27, P53, P57, GAPDH, H2A, METTL3, METTL14, WTAP, ALKBH5, LIN28, L-MYC The amplification primers of 25 genes including CDX2 and CDX2 were mixed in proportion to make a primer pool, and the respective final concentrations of the amplification primers of the 25 genes were 0.1 μM.

[0060] S2: Configuration of RT-PreAmp Master Mix (Reverse Transcription Reaction System)

[0061] In 4 nucleic acid-free centrifuge tubes, configure 5.0 μl of RT-PreAmp Master Mix (reverse transcription reaction system) and place them on ice for later use. Then take porcine oocytes with good morphology and wash them with DPBS (Duchenne's phosphate Buffer) was washed three times, and one oocyte was added to each of the four centrifuge tubes, immediately pla...

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PUM

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Abstract

The invention discloses a method for quantitatively expressing a gene of a porcine single oocyte. The method comprises the following steps: S1: preparing a primer pool; S2: preparing a reverse transcriptional reaction system; S3: carrying out PCR (Polymerase Chain Reaction); S4: diluting template DNA (Deoxyribonucleic Acid); S5: carrying out qPCR (quantitative Polymerase Chain Reaction) and the like, so as to realize quantitative expression of the gene of the porcine single oocyte. According to the method disclosed by the invention, single cell quantitative PCR is adopted, and RNA (RibonucleicAcid) extraction and purification, reverse transcription and PCR are finished in the same tube, and extra operation is not needed; the method has the advantages of reducing testing errors, reducing the pollution, improving the sensitivity and the like; the quantitative PCR is carried out by adopting the single cell, so that the demanded quantity of samples is greatly reduced, the time is shortened, the vigor is reduced, and the cost is reduced; meanwhile, the limitation of a quantitative PCR technology is also overcome when the quantitative PCR technology is applied to precious samples. The method disclosed by the invention has the advantages of simplicity in operation, low cost, small errors, high accuracy and good effect.

Description

technical field [0001] The invention relates to the field of cell biology, in particular to a method for quantitatively expressing genes in a single porcine oocyte. technical background [0002] Nowadays, the development of animal embryo engineering technology has greatly promoted the progress of animal husbandry and biomedicine. It can be used to improve livestock breeds, breed new transgenic varieties, prepare human disease models, produce medicinal proteins, and study the mechanism of embryo development. Pigs are a top priority for embryonic biologists and geneticists because their physiology and anatomy share strong similarities with humans. [0003] In animal husbandry production, low sow fecundity is one of the key factors affecting production efficiency and economic benefits, and early embryonic death is considered to be one of the main reasons for sow fecundity decline, and about 30% of embryonic deaths occur The period between fertilized egg formation and embryo im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6851
CPCC12Q1/6806C12Q1/6851C12Q2531/113C12Q2521/107C12Q2563/107
Inventor 曹祖兵许腾腾高迪童旭张丹丹王怡青张运海
Owner ANHUI AGRICULTURAL UNIVERSITY