Screening and identifying method of miRNAs (micro Ribonucleic Acids) of fetal fibroblasts of Saanen dairy goats

A technology of fibroblasts, Saanen milk goats, applied in the field of biology

A technology of fibroblasts, Saanen milk goats, applied in the field of biology

CN106520758AInactive Publication Date: 2017-03-22TARIM UNIV
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  • Screening and identifying method of miRNAs (micro Ribonucleic Acids) of fetal fibroblasts of Saanen dairy goats
  • Screening and identifying method of miRNAs (micro Ribonucleic Acids) of fetal fibroblasts of Saanen dairy goats
  • Screening and identifying method of miRNAs (micro Ribonucleic Acids) of fetal fibroblasts of Saanen dairy goats

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Embodiment Construction

[0044] The technical solution of the present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments.

[0045] A screening and identification method of Saanen dairy goat fetal fibroblast miRNA, comprising the following steps:

[0046] Step 1. Obtaining Saanen dairy goat fetal fibroblasts:

[0047] Select 5 dairy goats for simultaneous estrus, check the pregnancy status by B-ultrasound about 30 days after the last mating, select a successful pregnancy and take out the fetus aseptically by surgery at about 40-45 days, wash it with PBS containing double antibodies three times, put Into the serum-free medium quickly brought back to the laboratory. Aseptically remove the head, limbs and viscera in the cell chamber, rinse the remaining skin tissue three times with alcohol and PBS three times, and then cut into 1mm 3 Paste the small pieces on the cell culture dish for culture, change the medium every three days, dig...

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Abstract

The invention discloses a screening and identifying method of miRNAs (micro Ribonucleic Acids) of fetal fibroblasts of Saanen dairy goats. The method comprises the following steps: obtaining the fetal fibroblasts of the Saanen dairy goats; extracting RNAs; constructing a library and carrying out high-throughput sequencing; and carrying out data processing. By adopting the high-throughput sequencing, 16395039total_reads are obtained, and redundant data is taken out to obtain clean_reads 16150181 containing Unique sRNAS 205857. Bioinformatic analysis is carried out to obtain 44 known miRNAs and 247 candidate new miRNAs. The quantity of miRNA target genes is 5401, and the quantity of sites of the target genes is 6069; and the quantity of candidate miRNA target genes is 8401, and the quantity of sites of the target genes is 10832.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for screening and identifying miRNA in Saanen dairy goat fetal fibroblast cells. Background technique [0002] So far, there are three main methods for the identification and discovery of miRNAs: miRNA cDNA library sequencing, bioinformatics prediction and forward genetic screening. [0003] Bioinformatics has discovered a large number of new miRNAs, but this method has a drawback: it identifies new miRNAs by summarizing known miRNA sequences or precursor sequences, which leads to low accuracy of prediction results and is prone to false positives. positives and false negatives. Forward genetic screening is a research method initially used by molecular biologists, which identifies candidate miRNAs through the variant phenotypes produced by spontaneous mutations or artificial mutagenesis of individual organisms or cell genomes. Using this method, lin-4 and let-7 ...

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Application Information

Patent Timeline
22 Mar 2017
Publication
CN106520758A
IPC
C12N15/10; C40B50/06; C12Q1/68
CPC
C12N15/1003; C12Q1/6869; C12Q2535/122; C40B50/06; C12Q2525/191
Inventors
常卫华; 武军元