CN104630222B - A kind of miRNA and the specific primer being detected to it and method - Google Patents
A kind of miRNA and the specific primer being detected to it and method Download PDFInfo
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- CN104630222B CN104630222B CN201410740648.2A CN201410740648A CN104630222B CN 104630222 B CN104630222 B CN 104630222B CN 201410740648 A CN201410740648 A CN 201410740648A CN 104630222 B CN104630222 B CN 104630222B
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Abstract
The present invention relates to a kind of miRNA (miR n 789), the mature sequence of the miRNA has SEQ ID NO:Nucleotide sequence shown in 1.Present invention also offers the specific primer and detection method of the fluorescence quantitative PCR detection miRNA.MiRNA provided by the present invention can be applied to animal molecular genetics field.The transcriptional level of miRNA provided by the present invention can be carried out using above-mentioned primer provided by the present invention and method fast, accurately quantitative.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of miRNA and the specific primer that is detected to it and
Method.
Background technology
MiRNA (microRNA) is the non-coding RNA of about 20~23 nucleotide of one group of length by genome encoding, class
The molecule of siRNA is similar to, by guiding silencing complex (RISC) degraded mRNA with target gene mRNA base pairings or hindering it
Translation.MiRNAs is quite conservative in spore, and the miRNAs found in plant, animal and fungi is only specifically being organized
Expressed with the stage of development, miRNA tissue specificities and timing determine the specific Function of tissue and cell, show miRNA
Serve during the adjusting of cell growth and growth course a variety of.
Although some miRNA are found that in organism at present, but researcher still knows it to the function of miRNA
It is very few.
The present inventor has found during studying sheep seasonal oestrus character, the regulation and control of miRNA
May be related with the reproductive status of sheep.
Therefore, it is necessary to be separated to the miRNA of unknown participation sheep reproductive status regulation and control, and what is more important is built
The quantitative detection primer and method of corresponding miRNA is found so as to understand its relation between sheep reproductive status.
The content of the invention
First purpose of the present invention is to provide a kind of miRNA.
To understand the relation between sheep reproductive status and miRNA regulation and control, the present inventor is from different reproductive status
The new miRNA of a differential expression is detected in lower Sheep Ovary tissue, is temporarily named as miR-n-789, its mature sequence tool
Just like SEQ ID NO:Nucleotide sequence shown in 1, its precursor sequence have such as SEQ ID NO:Nucleotide sequence shown in 2.
It yet there are no the report that fluorescent quantitative PCR detection method is carried out to miR-n-789 both at home and abroad at present.
In order to study transcriptional levels of the miR-n-789 in particular organization or cell, it is necessary to establish one kind can be to miR-
The method that n-789 carries out accurate quantification, and then lay the first stone for research miR-n-789 functions and its influence factor.Therefore, this hair
Second bright purpose is to provide a kind of specific primer good to miRNA (miR-n-789) detection result.
In order to reach second object of the present invention, the present invention provides the miRNA (miR- described in a kind of claim 1
N-789 specific primer), the nucleotides sequence of the primer are classified as：
Sense primer：5’—CGCTGGGCTGGAAGAA—3’；
Anti-sense primer：5’—TGGTGTCGTGGAGTCGG—3’.
Third object of the present invention is to provide a kind of accurate, easy-operating to miRNA progress fluorescence quantitative PCR detections
Method.
In order to reach third object of the present invention, fluorescence quantitative PCR detection is carried out to miRNA the present invention provides a kind of
Method.
Preferably, this method further includes,
Utilize SEQ ID No:Sequence shown in 5 carries out reverse transcription to total serum IgE for reverse transcription primer and obtains template cDNA.
Preferably, the Total RNAs extraction is from Sheep Ovary tissue.
Preferably, the fluorescence quantitative PCR detection is used as reference gene using U6 snRNA.
Preferably, the working reaction system of the quantitative fluorescent PCR is:
Preferably, the working procedure of the quantitative fluorescent PCR is：95 DEG C of pre-degeneration 30s；95 DEG C of denaturation 5s, 60 DEG C of annealing
34s, 40 circulations.
Preferably, the described method comprises the following steps：
The acquisition of total serum IgE：Separated, precipitated by RNA after flesh tissue is homogenized, rinsed, the dry and molten step of weight obtains
Total serum IgE；
Reverse transcription：Utilize SEQ ID No:Sequence shown in 5 carries out reverse transcription to total serum IgE for reverse transcription primer and obtains template
cDNA；
Quantitative fluorescent PCR：Sense primer, anti-sense primer, ROX are added after template cDNA, SYBR TaqTM II is pre-mixed
Dyestuff II and sterile purified water are configured to the reaction system of 20 μ L, U6 snRNA are selected as reference gene, in 95 DEG C of pre-degenerations
30s；95 DEG C of denaturation 5s, 60 DEG C of annealing 34s, 40 circulate.
Present invention also offers the application of the miRNA (miR-n-789).
MiR-n-789 can be applied to animal molecular genetics field, utilize above-mentioned primer provided by the present invention and side
Method carries out the transcriptional level of miR-n-789 fast, accurately quantitative.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Brief description of the drawings
Fig. 1 is the amplification curve diagram of sheep known for its fine thick wool miR-n-789 quantitative fluorescent PCRs reaction.
Fig. 2 is the solubility curve figure of sheep known for its fine thick wool miR-n-789 quantitative fluorescent PCRs reaction.
Embodiment
The embodiment of the present invention will be described in detail below.It is it should be appreciated that described herein
Embodiment is merely to illustrate and explain the present invention, and is not intended to limit the invention.
On the one hand, the present invention provides a kind of miRNA, the mature sequence of the miRNA to have SEQ ID NO：Shown in 1
Nucleotide sequence.
In one embodiment of the invention, the miRNA is isolated from Mammalian Ovary tissue, it is preferred that separation
From Sheep Ovary tissue, it is more highly preferred to, the miRNA is isolated from the ovary tissue of sheep known for its fine thick wool.
" separation " used herein refers to that material is separated from its primal environment, and keeps its original biological characteristic
And state.The miRNA can be processed from miRNA precursor sequences, generally be cut into miRNA points of maturation in the cell
Son.
MiRNA is studied for convenience, the miRNA is named as miR-n-789 by the present inventor.
One aspect of the present invention provides the precursor sequence of miR-n-789, its precursor sequence has such as SEQ ID NO:2 institutes
The nucleotide sequence shown.MiRNA is processed by precursor miRNA, and precursor sequence can be folded into stable loop-stem structure.Institute
The precursor sequence stated can be natural or artificial synthesized.
The present inventor using FlashPAGE fractionator (Ambion, Life Technologies,
Paisley, UK) kit by length be less than 40 nucleotide RNA separated from total serum IgE, then utilize Illumina
TruSeq Small RNA Sample Preparation kit structure miRNA libraries, utilize Illumina Hiseq 2000
Microarray dataset carries out high-flux sequence to library, and a large amount of new miRNA are finally obtained from the ovary of sheep different reproductive state,
1 difference miRNAs of the miR-n-789 between anestrus and oestrus ovary.
In one aspect of the invention, there is provided a kind of specific primer of detection miRNA, the miRNA is miR-
N-789, the nucleotides sequence of the primer are classified as：
Sense primer：(such as SEQ ID No of 5 '-CGCTGGGCTGGAAGAA -3 ':Shown in 3) and anti-sense primer：5’—
(such as SEQ ID No of TGGTGTCGTGGAGTCGG -3 '：Shown in 4).
The primer that those skilled in the art can be provided according to the present invention, using this area conventional technology to mesh
Mark miRNA (miR-n-789) is expanded.
On the other hand, the present invention provides a kind of quantitative fluorescent PCR inspection is carried out using primer pair miRNA (miR-n-789)
The method of survey.
According to the present invention, the fluorescent quantitative PCR detection method further includes：Utilize SEQ ID No:Sequence shown in 5 is
Reverse transcription primer carries out total serum IgE reverse transcription and obtains cDNA templates, then carries out fluorescence using the primer as template using cDNA and determine
Measure PCR detections.
Wherein, the Total RNAs extraction is from Tissues of Sheep, and in the case of preferable, the total serum IgE is derived from the ovary group of sheep
Knit.
Method provided by the present invention has no particular limits for the extracting method of total serum IgE, can be normal according to this area
The operating method of rule carries out.
Reference gene must be set up during the sample of detection every time of the invention, it is preferred that the reference gene can be U6 snRNA,
(small nuclear RNA (small nuclear RNA, snRNA) is RNA spliceosomes during eucaryote transcription post-processing to U6snRNA
Main component, participates in the process of mRNA precursor, its property is stablized, the rich content in cell, and small snRNA is to cut in U6 cores
One of constituent element of junctor, participates in the process of mRNA precursor.
When by the use of U6 snRNA as reference gene, SEQ ID No can be utilized:Sequence shown in 6 is sense primer
SEQ ID No:Sequence shown in 7 expands U6snRNA for anti-sense primer.
When the fluorescent quantitative PCR reaction system of the present invention is 20 μ L reaction systems, its preferable configuration mode is:
The working procedure of quantitative fluorescent PCR of the present invention is：95 DEG C of pre-degeneration 30s；95 DEG C of denaturation 5s, 60 DEG C of annealing
34s, 40 circulations.
Specifically, the method for the invention comprises the following steps：
The acquisition of total serum IgE：Separated, precipitated by RNA after flesh tissue is homogenized, rinsed, the dry and molten step of weight obtains
Total serum IgE；
Reverse transcription：Utilize SEQ ID No:Sequence shown in 5 carries out reverse transcription to total serum IgE for reverse transcription primer and obtains template
cDNA；
Quantitative fluorescent PCR：Sense primer, anti-sense primer, ROX are added after template cDNA, SYBR TaqTM II is pre-mixed
Dyestuff II and sterile purified water are configured to the reaction system of 20 μ L, U6snRNA are selected as reference gene, in 95 DEG C of pre-degenerations
30s；95 DEG C of denaturation 5s, 60 DEG C of annealing 34s, 40 circulate.
Method provided by the present invention can also calculate the relative expression quantity of miR-n-789 using formula one.
Formula one：Relative expression quantity=2 of miR-n-789-(CTmiRNA-CTcontrol)
In formula one, CTmiRNA is the cycle threshold of miR-n-789；CTcontrol is the circulation threshold of reference gene
Value, when the reference gene is U6snRNA, CTcontrol CTU6snRNA.
Present invention also offers the application of the miR-n-789, the application includes but not limited in Sheep Ovary tissue
The quantitative analysis of miR-n-789.
The content of one's duty invention is further illustrated by the following examples.Unless otherwise specified, skill used in embodiment
The conventional means that art means are well known to those skilled in the art.
Embodiment 1
Fresh sheep known for its fine thick wool ovary tissue is taken, is ground in liquid nitrogen, carries out RNA separation, extracts total serum IgE.Utilize FlashPAGE
Length is less than 40 nucleotide by fractionator (Ambion, Life Technologies, Paisley, UK) kits
RNA is separated from sheep known for its fine thick wool total serum IgE, then utilizes Illumina TruSeq Small RNA Sample Preparation
Kit structure miRNA libraries, carry out high-flux sequence, finally from beach using 2000 microarray datasets of Illumina Hiseq to library
Obtain a large amount of new miRNA in the ovary of sheep different reproductive state, miR-n-789 between anestrus and oestrus ovary 1
A difference miRNA.Its mature sequence has SEQ ID NO:Nucleotide sequence shown in 1.
Embodiment 2
(1) Total RNAs extraction, step are as follows：
Homogenate：Take fresh sheep known for its fine thick wool ovary tissue (totally two groups, every group of 3 samples, wherein, first group of 3 samples are derived from
3 anestrus grow up sheep known for its fine thick wools, and second group of 3 samples are derived from 3 oestrus adult sheep known for its fine thick wools, and two groups of sheep known for its fine thick wools are of the same age and weight differences
In 1KG.), after carrying out tissue homogenate using the mortar by DEPC processing and Liquid nitrogen precooler, it is transferred in the EP pipes of 1.5mL,
Add 1mL TRIPURE reagents, shaking a moment.
The separation of RNA：Homogenised sample 5min is incubated, nucleic acid is fully cracked separate out, every milliliter of TRIPURE reagent adds
Add 0.2mL chloroforms, cover bottle cap, rock EP pipes 15sec at full tilt with hand, 3min, 1200g centrifugations 15min (4 are placed at 25 DEG C
DEG C) layering：Lower floor is phenol-chloroform, and upper strata is water phase, and RNA is present in water phase.Water phase volume should be the 60% of cumulative volume.
The precipitation of RNA：Upper strata aqueous phase is drawn in the EP pipes of a new 1.5mL, leaves organic phase.With isopropanol precipitating RNA,
Ratio with being most initially added into TRIPURE amounts is 2:1,25 DEG C of placement 10min, 1200g centrifugation 10min (4 DEG C).
The drop of similar gels can be formed after RNA centrifugations in tube bottom or side wall.
RNA is rinsed：Supernatant is removed, RNA precipitate is rinsed with 75% ethanol, the ratio with being most initially added into TRIPURE amounts is 1:
1.Concussion mixes sample, and 2 DEG C~8 DEG C 7500g centrifuge 5min.
Dry RNA：It is air-dried or is dried in vacuo 5~10min.
The molten RNA of weight：RNA is dissolved in DEPC water of the 20 μ L without RNase, under piping and druming is several, is placed in 55~60 DEG C of 10min,
It is dissolved as far as possible, it is stand-by to store in -70 DEG C of refrigerators.
(2) reverse transcription, step are as follows：
Reverse transcription is carried out in the 200 μ LPCR pipes of the processed no Rnase with DEPC.Operating process according to
PrimeScript RT reagent Kit (TaKaRa, Cat RR037A) specification carries out.The reverse transcription primer of miR-n-789
For SEQ ID No:Sequence shown in 5.
(3) quantitative fluorescent PCR reacts, and step is as follows：
20 μ L reaction systems：The cDNA of 2.0 μ L is (with 1:4 dilution) be used for expand, and with 10 μ L SYBR TaqTM II
(TaKaRa, Dalian, China) is pre-mixed, the miR specific primers group (each 0.8 μ L of forward and reverse primer) of 1.6 μ L, and 0.4
The water of the ROX dyestuffs II of μ L and 6 μ L.Amplification program：95 DEG C of 30s, then 95 DEG C of 5s, 60 DEG C of 34s, are circulated 40 times.Selection
U6snRNA is as reference gene.Utilize SEQ ID No:Sequence shown in 6 is sense primer SEQ ID No:Sequence shown in 7
U6snRNA is expanded for anti-sense primer.
(4) relative expression's variance analysis of gene：
The relative expression quantity of miR-n-789 is calculated using formula one.Draw amplification curve diagram (see Fig. 1) and solubility curve figure
(see Fig. 2).
Formula one：The relative expression quantity of miR-n-789=：2-(CTmiRNA-CTU6snRNA)
(5) result：
1) amplification curve and solubility curve of sheep miR-n-789 quantitative fluorescent PCRs reaction.
By the result of Fig. 1 and Fig. 2 it is known that all individual amplification curves and solubility curve are more consistent；In miR-
The rarely seen single peak of solubility curve that quantitative fluorescent PCR reacts in the amplification procedure of n-789, shows that the purpose fragment of amplification is special
Property is good.
2) difference of different reproductive state Sheep Ovary tissue miR-n-789 transcriptional levels.Pass through real-time fluorescence quantitative PCR
Method understand, different reproductive state Sheep Ovary tissue miR-n-789 transcriptional levels have significant difference (P<0.05).As a result
It is shown in Table 1.
Table 1
| Group | MiR-n-789 expression quantity (again) |
| Anestrus | 1a |
| Oestrus | 0.10±0.02b |
By result above as can be seen that using specific primer provided by the present invention, pass through fluorescence quantifying PCR method
The expression of miR-n-789 in tissue samples can fast, accurately be analyzed, obtain the sheep of different reproductive state
Relative expression's situation of miR-n-789 in ovary.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a kind of miRNA, it is characterised in that the mature sequence of the miRNA has SEQ ID NO:Nucleotides sequence shown in 1
Row.
2. the specific primer of the miRNA described in a kind of claim 1, it is characterised in that the nucleotides sequence of the primer is classified as：
Sense primer：5’—CGCTGGGCTGGAAGAA—3’；
Anti-sense primer：5’—TGGTGTCGTGGAGTCGG—3’.
3. the method for fluorescence quantitative PCR detection is carried out using miRNA described in the primer pair described in claim 2.
4. according to the method described in claim 3, it is characterized in that, the method further includes：
Utilize SEQ ID No:Sequence shown in 5 carries out reverse transcription to total serum IgE for reverse transcription primer and obtains template cDNA.
5. the method according to claim 3 or 4, it is characterised in that the working reaction system of the quantitative fluorescent PCR is：
6. according to the method described in claim 5, it is characterized in that, the working procedure of the quantitative fluorescent PCR is：95 DEG C of pre- changes
Property 30s；95 DEG C of denaturation 5s, 60 DEG C of annealing 34s, 40 circulate.
7. according to the method described in claim 6, it is characterized in that, the fluorescence quantitative PCR detection using U6 snRNA in
Join gene.
8. according to the method described in any one in claim 3,4,6 and 7, it is characterised in that the Total RNAs extraction is certainly continuous
Sheep ovary tissue.
9. according to the method described in any one in claim 3,4,6 and 7, it is characterised in that the described method includes following step
Suddenly：
The acquisition of total serum IgE：Separated, precipitated by RNA after flesh tissue is homogenized, rinsed, the dry and molten step of weight obtains always
RNA；
Reverse transcription：Utilize SEQ ID No:Sequence shown in 5 carries out reverse transcription to total serum IgE for reverse transcription primer and obtains template
cDNA；
Quantitative fluorescent PCR：Sense primer, anti-sense primer, ROX dyestuffs are added after template cDNA, SYBR TaqTM II is pre-mixed
II and sterile purified water be configured to the reaction system of 20 μ L, U6 snRNA are selected as reference gene, in 95 DEG C of pre-degeneration 30s；
95 DEG C of denaturation 5s, 60 DEG C of annealing 34s, 40 circulate.
10. applications of the miRNA in animal molecular genetics field described in claim 1.
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| CN114657182B (en) * | 2022-04-18 | 2023-09-19 | 中国农业科学院北京畜牧兽医研究所 | miRNA (micro ribonucleic acid) and specificity detection method and application thereof |
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| CN102409064A (en) * | 2011-03-23 | 2012-04-11 | 中国农业科学院北京畜牧兽医研究所 | Interference vector for improving animal fertility |
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| 深度测序鉴定绵羊microRNA转录组;张世芳等;《中国畜牧兽医》;20130930;第30卷(第9期);19-22页 * |
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