CN106520758A - Screening and identifying method of miRNAs (micro Ribonucleic Acids) of fetal fibroblasts of Saanen dairy goats - Google Patents
Screening and identifying method of miRNAs (micro Ribonucleic Acids) of fetal fibroblasts of Saanen dairy goats Download PDFInfo
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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
The invention belongs to biological technical field, specifically, is related to a kind of Sa energy milch goat fetal fibroblast miRNA
Screening and authentication method.
Background technology
Up to now, the discriminating of miRNA and discovery mainly has three kinds of methods：MiRNA cDNA library PCR sequencing PCRs, biological letter
Breath learns predicted method and forward genetics screening.
Bioinformatics has discovered that a large amount of new miRNA, but the method has a drawback：It is by known
The summary of miRNA sequence or precursor sequence rule goes to differentiate new miRNA, therefore the accuracy that causes to predict the outcome is not high, and
Easily there is false positive and false negative.Forward genetics screening is a kind of initially use research method of molecular biologist, it
It is variation phenotypes that spontaneous mutation by bion or cellular genome or induced mutations are produced and identifies candidate miRNA.
Using the method, identification is found that lin-4, let-7 earliest, and discriminating was found that the miR- in fruit bat again by this method later
278th, the lsy-6 in miR-14 and nematode.But, screened by forward genetics and differentiate that the efficiency comparison of candidate miRNA is low；
Undergo mutation, lack, misplacing or the change of base only makes phenotype that small difference occurs if miRNAs is single base
Even without change, cause the discriminating of many candidate miRNAs to occur to omit, therefore the method is slowly eliminated.
MiRNA cDNA library PCR sequencing PCRs include Direct Cloning method and high-flux sequence method, and Direct Cloning method efficiency compares
It is relatively low, waste time and energy, with obvious limitation.Relative to cloning and sequencing technology, high-flux sequence has high efficiency, accuracy
And the advantages of rapidity, only enter performing PCR amplification after extracting the connection reverse transcription of RNA joints, directly go up machine sequencing, it is not necessary to
Cloning procedure, then sequencing result is carried out into bioinformatic analysis and microRNA target prediction etc., so as to obtain new miRNA.At present, it is high
Flux sequencing technologies have been widely applied in the middle of the miRNA researchs of various plants and animal.At present, also do not have in prior art
A kind of screening of Sa energy milch goat fetal fibroblast miRNA and authentication method.
The content of the invention
It is an object of the invention to provide a kind of screening of Sa energy milch goat fetal fibroblast miRNA and identification side
Method, the method are entered to Sa energy milch goat fetal fibroblast miRNA using high throughput sequencing technologies and bioinformatics technique
Row identification simultaneously makees GO functional analyses and KEGG path analysises.
Its concrete technical scheme is：
A kind of screening of Sa energy milch goat fetal fibroblast miRNA and authentication method, comprise the following steps：
The acquisition of step 1, Sa energy milch goat fetal fibroblast：
Choose 5 milch goats and do estrus synchronization, with last time 30 days ultrasound diagnosis pregnancy status of breeding, select successful pregnancy
And passed through the aseptic taking-up fetus of modus operandi with 40-45 days, with rinsing three times containing dual anti-PBS, it is put into serum free medium fast speed belt
Go back to laboratory.In cell room it is aseptic except decaptitating, four limbs, internal organ, remaining skin histology alcohol rinse three times, PBS rinse three times,
Then it is cut into 1mm3Fritter is attached to Tissue Culture Dish and is cultivated, and changed a subculture per three days, disappears when cell is paved with 90%
Change is passed on, and makees growth curve and karyotyping；
Step 2, RNA are extracted：
Take the logarithm growth period cell with trizol methods extract RNA, all items that the experiment is used include 1000mL,
200 μ L, 10 μ L tip heads, pipette tips box, Enpendoff pipes must with 0.1% DEPC water or without RNase water soak more than 6h or
Directly overnight, rear autoclaving 20-30min, 65 DEG C of dry for standby are packaged；All mortars for using, glassware, metal device
Tool masking foil toasts more than 8h during 180 DEG C of baking ovens are put in after packaging, and Temperature fall is standby, micro point of the total serum IgE Jing of extraction
Light photometer checks purity and concentration, suitable to send Hua Da gene to carry out high-flux sequence later；
Step 3, library construction and high-flux sequence
Above-mentioned total serum IgE Jing PAGE glues purifying, reverse transcription are taken, and then high pass measurement are carried out by microarray dataset of HiSeq2000
Sequence construction cDNA library；
Step 4, data processing
Sa energy milch goat fetal fibroblast sample obtains redundant data after the sequencing of HiSeq2000 platforms, to the original
Beginning data first have to carry out low quality sequence, remove joint sequence and series processing of depolluting, and it is clean to obtain clean sequence
Reads, then carries out next step bioinformatic analysis again；
The clean reads of acquisition are carried out blast with sheep full-length genome database to compare, SOAPv1.11 softwares are used
Analytical sequence differential expression and distribution situation, in order that each unique sRNA has unique annotation, we are using following preferential
Rule：rRNAetc>known miRNA>repeat>exon>Intron, due to rRNAetc be Jing Genbank databases and
Rfam databases are compared and are obtained, and the priority between experiment two databases of regulation is Genbank>Rfam, no comparison take up an official post
The sRNA of what annotation information is represented with unann.
Further to analyze candidate's miRNA secondary structures, experiment Mfold3.2 softwares]With RNAfold online softwares
New miRNA neck ring structures and minimum free energy are predicted, each candidate's sequence are further analysed in depth with MIREAPv0.2 softwares
Row, parameter setting are as follows：
(1) miRNA sequence length is 18-26nt
(2) miRNA reference sequences length is 20-24nt
(3) Drosha/Dicer restriction enzyme sites are at least 3
(4) miRNA maximum copy numbers on reference sequences are 20
(5) the maximum free energy that miRNA precursors are allowed is 200kcal/mol
(6) between miRNA and miRNA*, maximum space number is 35
(7) minimum between miRNA and miRNA* is 14 with logarithm
(8) between miRNAand miRNA*, maximum bulge loop is 4.
Further, specific operation process foundation Small RNA Sample Preparation Kit in step 3
(Illumina, USA) kit specification step is carried out, and simple procedure is as follows：
(1) total serum IgE is separated by 15% poly- propionamide gel electrophoresis first, cutting fragment is 18-30nt parts, Ran Houjin
Row is isolated and purified, glue reclaim；
(2) by T4RNA ligase is respectively in 3 ' ends and 5 ' 3 ' ((PO of end addition4)-CTGTAGGCACCATCAA-(NH2)-
(CH2)6) and 5 ' (ATCGTAGGCACCUGAAA) joint sequence
(3) RNA for successfully being added joint sequence carries out reverse transcription into cDNA, and then 25 circulate into performing PCR amplification.
(4) pcr amplification product of 90bp or so its product Jing after PAGE glue purifying is reclaimed is scientific and technological in Shenzhen Hua Da
HiSeq2000 platforms are sequenced.
(5) fragment is sequenced and removes the laggard row information biology of low quality sequence, joint sequence, repetitive sequence and polluted sequence
Credit is analysed.
Further, low quality sequence is carried out in step 4, removes joint sequence and the process of series processing of depolluting is concrete
It is as follows：
(1) remove the reads for there are 5 ' joint sequences；
(2) remove the reads for not having 3 ' joint sequences；
(3) remove the relatively low reads of sequencing quality；
(4) remove the reads for being not inserted into fragment；
(5) remove the reads containing polyA；
(6) remove the fragment less than 18 nucleotides.
Compared with prior art, beneficial effects of the present invention：
The present invention obtains 16395039 total_reads by high-flux sequence, obtains after taking out redundant data
Clean_reads 16150181, wherein Unique sRNAS 205857.Bioinformatic analysis obtain known miRNA 44
Bar, the new miRNA247 bars of candidate.Known miRNA target genes quantity 5401, target gene number of sites are 6069；Candidate's miRNA target bases
Factor amount 8401, target gene site numerical digit 10832.
Description of the drawings
Fig. 1 is：High-flux sequence prepares flow chart；
Fig. 2 is：Bioinformatic analysis flow chart；
Fig. 3 is：The new miRNA precursors secondary structure of part candidate.
Specific embodiment
Technical scheme is described in more detail with specific embodiment below in conjunction with the accompanying drawings.
A kind of screening of Sa energy milch goat fetal fibroblast miRNA and authentication method, comprise the following steps：
The acquisition of step 1, Sa energy milch goat fetal fibroblast：
Choose 5 milch goats and do estrus synchronization, with last time 30 days or so ultrasound diagnosis pregnancy status of breeding, work(of hanking
Gestation simultaneously passed through the aseptic taking-up fetus of modus operandi with 40-45 days or so, with rinsing three times containing dual anti-PBS, was put into free serum culture
Base quickly takes back laboratory.In cell room it is aseptic except decaptitating, four limbs, internal organ, remaining skin histology alcohol rinse three times, PBS
Rinse three times, be then cut into 1mm3Fritter is attached to Tissue Culture Dish and is cultivated, and changes a subculture per three days, treats that cell is paved with
Had digestive transfer culture when 90%, and make growth curve and karyotyping；
Step 2, RNA are extracted：
Take the logarithm growth period cell with trizol methods extract RNA, all items that the experiment is used include 1000mL,
200 μ L, 10 μ L tip heads, pipette tips box, Enpendoff pipes etc. must with 0.1% DEPC water or without RNase water soak 6h with
Above or directly overnight, rear autoclaving 20-30min, 65 DEG C of dry for standby are packaged；All mortars for using, glassware, gold
Category apparatus etc. toasts more than 8h during 180 DEG C of baking ovens are put in after being packaged with masking foil, and Temperature fall is standby, the total serum IgE Jing of extraction
Micro-spectrophotometer checks purity and concentration, suitable to send Hua Da gene to carry out high-flux sequence later；
Step 3, library construction and high-flux sequence
Above-mentioned total serum IgE Jing PAGE glues purifying, reverse transcription are taken, and then high pass measurement are carried out by microarray dataset of HiSeq2000
Sequence construction cDNA library, builds storehouse process and bioinformatic analysis flow process as shown in Figure 1, Figure 2；
Specific operation process is according to Small RNA Sample Preparation Kit (Illumina, USA) kits
Specification step is carried out, and simple procedure is as follows：
(1) total serum IgE is separated by 15% poly- propionamide gel electrophoresis first, cutting fragment is 18-30nt parts, Ran Houjin
Row is isolated and purified, glue reclaim；
(2) by T4RNA ligase is respectively in 3 ' ends and 5 ' 3 ' ((PO of end addition4)-CTGTAGGCACCATCAA-(NH2)-
(CH2)6) and 5 ' (ATCGTAGGCACCUGAAA) joint sequence
(3) RNA for successfully being added joint sequence carries out reverse transcription into cDNA, and then 25 circulate into performing PCR amplification.
(4) pcr amplification product of 90bp or so its product Jing after PAGE glue purifying is reclaimed is scientific and technological in Shenzhen Hua Da
HiSeq2000 platforms are sequenced.
(5) fragment is sequenced and removes the laggard row information biology of low quality sequence, joint sequence, repetitive sequence and polluted sequence
Credit is analysed.
Step 4, data processing
Sa energy milch goat fetal fibroblast sample obtains redundant data after the sequencing of HiSeq2000 platforms, to the original
Beginning data first have to carry out low quality sequence, remove joint sequence and sequence of depolluting etc. is processed, and obtaining clean sequence is
Clean reads, then carry out next step bioinformatic analysis again.Its processing procedure is specific as follows：
(1) remove the reads for there are 5 ' joint sequences；
(2) remove the reads for not having 3 ' joint sequences；
(3) remove the relatively low reads of sequencing quality；
(4) remove the reads for being not inserted into fragment；
(5) remove the reads containing polyA；
(6) remove the fragment less than 18 nucleotides；
By clean reads and sheep full-length genome database (the International Sheep Genomics for obtaining
Consortium：ISGC blast comparisons) are carried out, with SOAPv1.11 software analysis sequence differential expressions and distribution situation, in order to
Each unique sRNA is made to have unique annotation, we adopt following priority rule：rRNAetc>known miRNA>repeat
>exon>Intron, as rRNAetc is that Jing Genbank databases and Rfam databases are compared and obtained, experiment two numbers of regulation
It is Genbank according to the priority between storehouse>Rfam, does not have the sRNA for comparing upper any annotation information to be represented with unann.
Further to analyze candidate's miRNA secondary structures, experiment Mfold3.2 softwares]With RNAfold online softwares
New miRNA neck ring structures and minimum free energy are predicted, each candidate's sequence are further analysed in depth with MIREAPv0.2 softwares
Row, parameter setting are as follows：
(1) miRNA sequence length is 18-26nt
(2) miRNA reference sequences length is 20-24nt
(3) Drosha/Dicer restriction enzyme sites are at least 3
(4) miRNA maximum copy numbers on reference sequences are 20
(5) the maximum free energy that miRNA precursors are allowed is 200kcal/mol
(6) between miRNA and miRNA*, maximum space number is 35
(7) minimum between miRNA and miRNA* is 14 with logarithm
(8) between miRNA and miRNA*, maximum bulge loop is 4.
The above, preferably specific embodiment only of the invention, protection scope of the present invention not limited to this are any ripe
Those skilled in the art are known in the technical scope of present disclosure, the letter of the technical scheme that can be become apparent to
Altered or equivalence replacement are each fallen within protection scope of the present invention.
Claims (3)
1. a kind of screening of Sa energy milch goat fetal fibroblast miRNA and authentication method, it is characterised in that including following step
Suddenly：
The acquisition of step 1, Sa energy milch goat fetal fibroblast：
Choose 5 milch goats do estrus synchronization, with last time breed 30 days ultrasound diagnosis pregnancy status, select successful pregnancy and with
By the aseptic taking-up fetus of modus operandi, with rinsing three times containing dual anti-PBS, it is put into serum free medium and quickly takes back reality within 40-45 day
Test room；In cell room it is aseptic except decaptitating, four limbs, internal organ, remaining skin histology alcohol rinse three times, PBS rinse three times, then
It is cut into 1mm3Fritter is attached to Tissue Culture Dish and is cultivated, and changes a subculture per three days, and digestion when cell is paved with 90% is passed
Generation, and make growth curve and karyotyping；
Step 2, RNA are extracted：
Take the logarithm growth period cell with trizol methods extract RNA, all items that the experiment is used include 1000mL, 200 μ L,
10 μ L tip heads, pipette tips box, Enpendoff pipes must soak more than 6h or direct mistakes with 0.1% DEPC water or without RNase water
At night, package rear autoclaving 20-30min, 65 DEG C of dry for standby；All mortars for using, glassware, metallic weapon tin
Foil paper toasts more than 8h during 180 DEG C of baking ovens are put in after packaging, and Temperature fall is standby, the micro spectrophotometrics of total serum IgE Jing of extraction
Meter checks purity and concentration, suitable to send Hua Da gene to carry out high-flux sequence later；
Step 3, library construction and high-flux sequence
Above-mentioned total serum IgE Jing PAGE glues purifying, reverse transcription are taken, and then high-flux sequence structure are carried out by microarray dataset of HiSeq2000
Build cDNA library；
Step 4, data processing
Sa energy milch goat fetal fibroblast sample obtains redundant data after the sequencing of HiSeq2000 platforms, to the original number
According to first having to carry out low quality sequence, joint sequence is removed and series processing of depolluting, it is clean to obtain clean sequence
Reads, then carries out next step bioinformatic analysis again；
The clean reads of acquisition are carried out blast with sheep full-length genome database to compare, SOAPv1.11 software analysis are used
Sequence differential expression and distribution situation, in order that each unique sRNA has unique annotation, we are preferentially advised using following
Then：rRNAetc>known miRNA>repeat>exon>Intron, as rRNAetc is Jing Genbank databases and Rfam
Database is compared and is obtained, and the priority between experiment two databases of regulation is Genbank>Rfam, without the upper any note of comparison
The sRNA for releasing information is represented with unann；
Further to analyze candidate's miRNA secondary structures, new miRNA is predicted with Mfold3.2 softwares and RNAfold online softwares
Neck ring structure and minimum free energy, further analyse in depth each candidate sequence, parameter setting with MIREAPv0.2 softwares
It is as follows：
(1) miRNA sequence length is 18-26nt
(2) miRNA reference sequences length is 20-24nt
(3) Drosha/Dicer restriction enzyme sites are at least 3
(4) miRNA maximum copy numbers on reference sequences are 20
(5) the maximum free energy that miRNA precursors are allowed is 200kcal/mol
(6) between miRNA and miRNA*, maximum space number is 35
(7) minimum between miRNA and miRNA* is 14 with logarithm
(8) between miRNAand miRNA*, maximum bulge loop is 4.
2. the screening of Sa energy milch goat fetal fibroblast miRNA according to claim 1 and authentication method, its feature
It is that specific operation process is according to Small RNASample Preparation Kit (Illumina, USA) reagents in step 3
Box specification step is carried out, and simple procedure is as follows：
(1) total serum IgE is separated by 15% poly- propionamide gel electrophoresis first, cutting fragment is 18-30nt parts, is then carried out point
From purifying, glue reclaim；
(2) by T4RNA ligase is respectively in 3 ' ends and 5 ' 3 ' ((PO of end addition4)-CTGTAGGCACCATCAA-(NH2)-
(CH2)6) and 5 ' (ATCGTAGGCACCUGAAA) joint sequence
(3) RNA for successfully being added joint sequence carries out reverse transcription into cDNA, and then 25 circulate into performing PCR amplification；
(4) pcr amplification product of 90bp or so Jing PAGE glue purifying reclaim after its product in Shenzhen Hua Da science and technology HiSeq2000
Platform is sequenced；
(5) fragment is sequenced and removes the laggard row information biology credit of low quality sequence, joint sequence, repetitive sequence and polluted sequence
Analysis.
3. the screening of Sa energy milch goat fetal fibroblast miRNA according to claim 1 and authentication method, its feature
It is in step 4, to carry out low quality sequence, removes joint sequence and the process of series processing of depolluting is specific as follows：
(1) remove the reads for there are 5 ' joint sequences；
(2) remove the reads for not having 3 ' joint sequences；
(3) remove the relatively low reads of sequencing quality；
(4) remove the reads for being not inserted into fragment；
(5) remove the reads containing polyA；
(6) remove the fragment less than 18 nucleotides.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108733974A (en) * | 2017-04-21 | 2018-11-02 | 胤安国际（辽宁）基因科技股份有限公司 | A kind of mtDNA sequence splicing and copy number method for measuring based on high-flux sequence |
| CN111793671A (en) * | 2020-07-22 | 2020-10-20 | 中国农业大学 | Optimized preparation method of chromosome suspension suitable for binary flow sorting of sheep chromosomes |
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| CN1500384A (en) * | 2002-11-15 | 2004-06-02 | 上海杰隆生物工程股份有限公司 | Method for promoting development rate of nucleus-transplanted early embryo of mammalian |
| CN104762265A (en) * | 2015-04-22 | 2015-07-08 | 扬州大学 | Novel method for obtaining goat-induced multi-potent stem cells |
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| 袁超: "绒山羊次级毛囊周期性变化相关 microRNA 的鉴定及 miR-125b在毛乳头细胞中的功能研究", 《中国博士学位论文全文数据库（电子期刊）农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108733974A (en) * | 2017-04-21 | 2018-11-02 | 胤安国际（辽宁）基因科技股份有限公司 | A kind of mtDNA sequence splicing and copy number method for measuring based on high-flux sequence |
| CN108733974B (en) * | 2017-04-21 | 2021-12-17 | 胤安国际（辽宁）基因科技股份有限公司 | Mitochondrial sequence splicing and copy number determination method based on high-throughput sequencing |
| CN111793671A (en) * | 2020-07-22 | 2020-10-20 | 中国农业大学 | Optimized preparation method of chromosome suspension suitable for binary flow sorting of sheep chromosomes |
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