CN106757380A - A kind of method for building pre miRNA3`RACE seq libraries in plant - Google Patents

A kind of method for building pre miRNA3`RACE seq libraries in plant Download PDF

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Publication number
CN106757380A
CN106757380A CN201710051734.6A CN201710051734A CN106757380A CN 106757380 A CN106757380 A CN 106757380A CN 201710051734 A CN201710051734 A CN 201710051734A CN 106757380 A CN106757380 A CN 106757380A
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dna
plant
primer
rna
seq
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CN106757380B (en
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宋剑波
岳路明
莫小为
杨海奇
刘琳
崔洁
陈雪梅
莫蓓莘
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Shenzhen University
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Abstract

The invention provides the method that one kind builds the RACE seq libraries of pre miRNA 3 ' in plant, the situation of change of the terminal bases of pre miRNA 3 ' in plant can be clearly seen using the method.The extraction that the present invention passes through RNA, adjunction head, reverse transcription, the methods such as nest-type PRC are carried out by special primer, successfully solve in plant, the problem of the structure hardly possible in the RACE seq libraries of pre miRNA 3 ', has founded the new method of the RACE seq library constructions of pre miRNA 3 ' for high-flux sequence in plant.

Description

The method that one kind builds the RACE-seq of pre-miRNA 3 ' libraries in plant
Technical field
The present invention relates to the method that one kind builds the RACE-seq of pre-miRNA 3 ' libraries in plant.
Background technology
With the development of sequencing technologies, the sequencing of transcript profile and degraded group is increasingly universal, used as the base of two generation sequencing technologies Plinth, the structure in library plays decisive role to sequencing result.The structure in a variety of RNA libraries has been occurred in that currently on the market It is very ripe, but in plant, the library constructing method to the RACE-seq of pre-miRNA 3 ' is not yet reported that.Pre- MiRNA as miRNA precursor, be the important intermediate in miRNA process, its 3 ' section modify for ripe miRNA Formation it is most important, so verifying the formation of the situation of change of the nucleotides of 3 ' pre-miRNA for studying miRNA has weight The biological significance wanted.However, because the abundance of pre-miRNA in plant is very low, length is (100bp-600bp) more long, and nothing Ploy (A) structure, ready-made banking process cannot all be applied to the structure in the RACE-seq of pre-miRNA 3 ' libraries.
The content of the invention
For above-mentioned weak point, the present invention provides a kind of side for building the RACE-seq of pre-miRNA 3 ' libraries in plant Method, this method carries out the separation of RNA using SDS-PAGE, adds 3 ' end connectors, and reverse transcription successfully constructs pre-miRNA 3 ' RACE-seq libraries.
The technology used in the present invention is as follows：The method that one kind builds the RACE-seq of pre-miRNA 3 ' libraries in plant, Including as follows：
(1), the extraction of plant total serum IgE；
(2), the preparation of RNA：
The plant total serum IgE for being extracted is separated using SDS-PAGE, the RNA between 50bp-400bp is carried out to cut glue Reclaim；
(3), connect：
The length of the RNA for having reclaimed concentrates on 50bp-400bp, connects a kind of special in the 3 ' ends of RNA by ligase DNA joints, sequence is as shown in sequence table SEQ ID No.1；
(4), reverse transcription：
Using reverse transcription reagent box, the RNA to having added good joint carries out reverse transcription, and reverse transcription is into cDNA；
(5), nest-type PRC reaction：
Using the special primer at 5 ' ends, the reverse transcription cDNA with step (4) carries out first round PCR as template；PCR primer After running PAGE glue recovery, recycling second to take turns primer carries out the second wheel PCR, and PCR primer can send to sequencing after reclaiming.
The present invention also has following technical characteristic：As above the first round PCR described in step (5), upper primer such as sequence table SEQ Shown in ID No.2-SEQ ID No.79, lower primer is as shown in sequence table SEQ ID No.80；Second wheel PCR, upper primer such as sequence Shown in list SEQ ID No.81, lower primer is as shown in sequence table SEQ ID No.82.
This method cleverly solves problem above using the special design of primers at the ends of pre-miRNA 5 ', constructs The high-quality RACE-seq of pre-miRNA 3 ' libraries.This method is a kind of using SDS-PAGE RNA isolation technics, 3 '-RACE Plus the technology such as joint technique and nest-type PRC, it is special and comprehensively build 3 ' pre-miRNA by the special design of primers at 5 ' ends The method in library, the 3 ' ends of pre-miRNA in plant can be analyzed in difference using the technology by two generation high throughput sequencing technologies Gene mutation under situation of change
Brief description of the drawings
Fig. 1 is Library development flow sketch of the present invention.
Specific embodiment
The present invention is further explained below according to specification citing：
Embodiment 1
As shown in figure 1, a kind of method for building the RACE-seq of pre-miRNA 3 ' libraries in plant, step is as follows
First, the extraction of plant total serum IgE
1st, the fresh plant tissues of 100mg are taken in clean 1.5mL centrifuge tubes.
2nd, it is plant tissue is quick-frozen in liquid nitrogen, smashed to pieces with pipette tips.
3rd, add 1mL TRIZOL to turn upside down mixing, 5min is stood at room temperature.
4th, 200 μ L chloroforms are added, is overturned and is mixed, be stored at room temperature 15min.
5th, 12000X g centrifugations 15min, draws the μ L of supernatant 400.
6th, 400 μ L isopropanols are added, is overturned and is mixed, -20 DEG C stand 30min shallow lake RNA.
7th, 4 DEG C, 12000X g centrifugation 15min abandon supernatant.
8th, the ethanol washing RNA of 1mL 75%, 4 DEG C, 12000X g centrifugations 5min are added.
9th, drying at room temperature RNA, adds 30 μ L ddH2O dissolves RNA.
10th, the RNA of extraction is deposited in into -80 DEG C of preservations.
2nd, the preparation (50-400) of RNA
1st, 15% urea-PAGE glue, common 15mL are configured：
Glue is poured into clamping plate, comb, gel at least 30min is put.
2nd, in the total serum IgE of 30 μ L, 2 × small RNA loading dye of equivalent are added, 70 DEG C of denaturation after mixing 5min, is placed on ice immediately after.
2×small RNA loading dye：80%formamide (formamide)；0.1%Xylene FF (dimethylbenzene FF)；0.1%Brophenol Blue (bromophenol blue)
3rd, point sample, and add DNA marker, the 150V electrophoresis 1-1.5h of 10 μ L 10bp.(Running buffer:0.5 ×TBE)
4th, EB dyeing 5min.
5th, glue is cut, the glue of 50-400bp is reclaimed to 1.5mL centrifuge tubes, and glue is consulted broken with the pipette tips of 1mL.
6th, 500 μ L 0.4N NaCl-DEPC are added to centrifuge tube, and ensures that glue can flow when centrifuge tube is overturn It is dynamic.
7th, centrifuge tube upset 6h or overnight is held for 4 DEG C.
8th, 4 DEG C, be transferred to supernatant in new centrifuge tube by 13200r/m centrifugation 1min.Repeat 2-3 times, until removal All of glue.
9th, 1 μ L Glycogen (glycogen) are added, 50 100% ethanol of μ L NaOAc and 1mL are put into -20 DEG C of 6h after mixing Or overnight.
10th, it is centrifuged and washs RNA with 70% ethanol.
11st, with 20 μ L DEPC- water dissolving RNAs, this RNA is used for building storehouse.
3rd, connect
(1) connect
The sequence of Linker is in above-mentioned linked system：
5′-rAppTGGAATTCTCGGGTGCCAAGG–NH2-3′；
Reaction condition：
1st, 25 DEG C of reaction 2h.
2nd, 65 DEG C of reaction 20min make enzyme denaturation.
(2) (50bp -420bp) is reclaimed
1st, 15% urea-PAGE glue is configured.
2nd, in the total serum IgE of 30 μ L, 2 × small RNA loading dye of equivalent are added, 70 DEG C of denaturation after mixing 5min, is placed on ice immediately after.
3rd, point sample, and add DNA marker, the 150V electrophoresis 1-1.5h of 10 μ L 10bp.
4th, EB dyeing 5min.
5th, glue is cut, the glue of 50-400bp or 50-250bp is reclaimed to 1.5mL centrifuge tubes, and glue is stabbed broken with 1mL pipette tips.
6th, 500 μ L 0.4N NaCl-DEPC are added to centrifuge tube, and ensures that glue can flow when centrifuge tube is overturn It is dynamic.
7th, 4 DEG C keep centrifuge tube upset 6h or overnight.
8th, 4 DEG C, be transferred to supernatant in new centrifuge tube by 13200r/m centrifugation 1min.Repeat 2-3 times, until removal All of glue.
9th, 1 μ L Glycogen (glycogen) are added, 50 100% ethanol of μ L NaOAc and 1mL are put into -20 DEG C of 6h after mixing Or overnight.
10th, it is centrifuged and the ethanol with 70% washes RNA, 20 μ L DEPC- water, -80 DEG C of preservations is dissolved in after drying.
4th, reverse transcription reaction
(1) reverse transcription
1. predegeneration
Ligated RNA 12μL
RT Primer 1μL
70 DEG C of reaction 10min, put rapidly 2min on ice.
2nd, reverse transcription reaction (20 μ L systems)
30 DEG C of 10min, 42 DEG C of 1h, after 70 DEG C of 15min denaturation, 4 DEG C of preservations.
(2) reclaim
12% native polyacrylamide gel reclaim the fragment of 50-420bp.
Prepare 12% PAGE glue (common 15mL)
1st, add 10 μ L 6 × DNA loading dye in PCR primer, all of DNA is clicked and entered into glue hole, with time point 10μL 10bp DNA marker。
2nd, gel electrophoresis 150V, 1.5h are carried out.
3rd, the DNA bands of 50-420bp are cut, DNA is reclaimed with the method for reclaiming tiny RNA.
4th, it is centrifuged, washing is dried.
5th, DNA is washed with 70% ethanol, 20 μ L DEPC- water, -80 DEG C of preservations is dissolved in after drying.
5th, nest-type PRC reaction
Two-wheeled PCR reacts (LA Taq)
(1) 10 circulations (20 μ L systems) of the first round
Primer：
Sense:1st PCR Forward primer (mix following 78 primers)
PCR reaction systems：
Reaction condition：98 DEG C of predegeneration 3min；98 DEG C of denaturation 10s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 10 are followed Ring；72 DEG C extend 5min, 12 DEG C of ∞ eventually.
(2) reclaim
12% native polyacrylamide gel reclaim the fragment of 50-200bp.
1st, 12% PAGE glue is prepared
2nd, add 10 μ L 6 × DNA loading dye in PCR primer, all of DNA is clicked and entered into glue hole, with time point 10μL 10bp DNA marker。
3rd, gel electrophoresis 150V, 1.5h are carried out；
4th, the DNA bands of 50-200bp are cut, DNA is reclaimed with the method for reclaiming tiny RNA；
5th, it is centrifuged, washing is dried；
6th, with the water dissolves DNA of 20-30 μ L.
(3) second wheel PCR (16 circulations)
Primer：
Sense:2nd PCR Forward primer：
AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA2nd PCR Anti-sense Anti-sense:
CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
PCR system：(20 μ L systems)
Reaction condition：98 DEG C of predegeneration 3min；98 DEG C of denaturation 10s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 16 are followed Ring；72 DEG C extend 5min, 12 DEG C of ∞ eventually.
(4) reclaim (fragment that 12% native polyacrylamide gel reclaim 100-300bp)
1st, 12% PAGE glue is prepared
2nd, add 10 μ L 6 × DNA loading dye in PCR primer, all of DNA is clicked and entered into glue hole, with time point 10μL 10bp DNA marker
3rd, gel electrophoresis 150V, 1.5h are carried out.
4th, the DNA bands of 100-300bp are cut, DNA is reclaimed with the method for reclaiming tiny RNA.
5th, it is centrifuged, washing is dried.
6th, with the water dissolves DNA of 20-30 μ L, it is used to be sequenced.
The ＞ Shenzhen University of ＜ 110
A kind of methods for building the RACE-seq of pre-miRNA 3 ' libraries in plant of the ＞ of ＜ 120
The ＞ 82 of ＜ 160
The ＞ 1 of ＜ 210
The ＞ 21 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ Linker of ＜ 213
The ＞ 1 of ＜ 400
TGGAATTCTC GGGTGCCAAG G
The ＞ 2 of ＜ 210
The ＞ 38 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR156a of ＜ 213
The ＞ 2 of ＜ 400
GAGTTCTACA GTCCGACGAT CCACAAAGGC AATTTGCA
The ＞ 3 of ＜ 210
The ＞ 44 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR156b of ＜ 213
The ＞ 3 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCGTCT ATAACTTTGC GTGT
The ＞ 4 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR156c of ＜ 213
The ＞ 4 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCGGCA CTTTGCATGT TCG
The ＞ 5 of ＜ 210
The ＞ 47 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR156d of ＜ 213
The ＞ 5 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCGGAA GTTGTATAAA AGTTTTG
The ＞ 6 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR156e of ＜ 213
The ＞ 6 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCCATG GTGGTTTCTT GCA
The ＞ 7 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR156f of ＜ 213
The ＞ 7 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCATGG TGGCTTTCTT GCA
The ＞ 8 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR156h of ＜ 213
The ＞ 8 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCGCAC AACCTGGGAT TAG
The ＞ 9 of ＜ 210
The ＞ 49 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR157a of ＜ 213
The ＞ 9 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCAGAT GATGAGATAC AATTCGGAG
The ＞ 10 of ＜ 210
The ＞ 49 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR157b of ＜ 213
The ＞ 10 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCCAGA TGATAAGATA CAATTCCTC
The ＞ 11 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR157c of ＜ 213
The ＞ 11 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCCTCT ACTCTTTTGT GCT
The ＞ 12 of ＜ 210
The ＞ 49 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR157d of ＜ 213
The ＞ 12 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCAAGA GCTAGAAGAC TATCTGCAT
The ＞ 13 of ＜ 210
The ＞ 49 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR158a of ＜ 213
The ＞ 13 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCCTTC TTTGTCTACA ATTTTGGAA
The ＞ 14 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR158b of ＜ 213
The ＞ 14 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTTGG AAAAGGTGAT GAT
The ＞ 15 of ＜ 210
The ＞ 45 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR159b of ＜ 213
The ＞ 15 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCAGCT TTCACTTACC CCTTT
The ＞ 16 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR160a of ＜ 213
The ＞ 16 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCGCCT GGCTCCCTGT ATG
The ＞ 17 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR160b of ＜ 213
The ＞ 17 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTGCC TGGCTCCCTG TAT
The ＞ 18 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR160c of ＜ 213
The ＞ 18 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCCCTG GCTCCCTGTA TGC
The ＞ 19 of ＜ 210
The ＞ 48 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR162a of ＜ 213
The ＞ 19 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCAATG TAAAAGCATG AATAGATC
The ＞ 20 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR162b of ＜ 213
The ＞ 20 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTGGA GGCAGCGGTT CAT
The ＞ 21 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR163 of ＜ 213
The ＞ 21 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCAGAG CACGGTCGAA GAA
The ＞ 22 of ＜ 210
The ＞ 47 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR164a of ＜ 213
The ＞ 22 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCCAAA CCAACAAACA CGAAATC
The ＞ 23 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR164b of ＜ 213
The ＞ 23 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCATGC GGAATTTTGT GAT
The ＞ 24 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR164c of ＜ 213
The ＞ 24 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTGGA GAAGCAGGGC ACG
The ＞ 25 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR165a of ＜ 213
The ＞ 25 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCGTTG TCTGGATCGA GGA
The ＞ 26 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR165b of ＜ 213
The ＞ 26 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCGCCA CATGGTATCG TCG
The ＞ 27 of ＜ 210
The ＞ 46 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR166a of ＜ 213
The ＞ 27 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTCTA ACAATCGAAT TGAACC
The ＞ 28 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR166b of ＜ 213
The ＞ 28 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCCTGG CTCGAGGACT CTT
The ＞ 29 of ＜ 210
The ＞ 44 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR166e of ＜ 213
The ＞ 29 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCCTCT TCTTTATTCA TTAG
The ＞ 30 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR166f of ＜ 213
The ＞ 30 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCGAAT GATGCCTGGC TCG
The ＞ 31 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR166g of ＜ 213
The ＞ 31 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTGGC TCGAGGTCAT GGA
The ＞ 32 of ＜ 210
The ＞ 45 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR167a of ＜ 213
The ＞ 32 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCCTTT CTTTATCCTT TGTTG
The ＞ 33 of ＜ 210
The ＞ 46 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR167c of ＜ 213
The ＞ 33 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCATAT TTCTTGTTCT TACAAG
The ＞ 34 of ＜ 210
The ＞ 45 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR168a of ＜ 213
The ＞ 34 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTTGG TTTGTGAGCA GGGAT
The ＞ 35 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR168b of ＜ 213
The ＞ 35 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTTGG CTGACACCGA CAC
The ＞ 36 of ＜ 210
The ＞ 44 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR169a of ＜ 213
The ＞ 36 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTTCC GTATAAAATA CAAG
The ＞ 37 of ＜ 210
The ＞ 49 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR169d of ＜ 213
The ＞ 37 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCACAA ATCTTAACTG ATTTTGGTG
The ＞ 38 of ＜ 210
The ＞ 47 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR169f of ＜ 213
The ＞ 38 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTTCA CAATCTGTTG ATTCGTG
The ＞ 39 of ＜ 210
The ＞ 45 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR169h of ＜ 213
The ＞ 39 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCGGTT GGTCGTCAGG CAGTC
The ＞ 40 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR169i of ＜ 213
The ＞ 40 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCATGA CCATTTTGCT TAT
The ＞ 41 of ＜ 210
The ＞ 45 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR169j of ＜ 213
The ＞ 41 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTGTT GAATCTTGCG GGTTA
The ＞ 42 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR169k of ＜ 213
The ＞ 42 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCAATA GACATCAGGC AGT
The ＞ 43 of ＜ 210
The ＞ 47 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR169m of ＜ 213
The ＞ 43 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCCTCA TCAAAAGACA TCAGGCA
The ＞ 44 of ＜ 210
The ＞ 45 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR169n of ＜ 213
The ＞ 44 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTGTT GAATCTTGCG GGTTA
The ＞ 45 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR170 of ＜ 213
The ＞ 45 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTGGC CTGGTTCACT CAG
The ＞ 46 of ＜ 210
The ＞ 49 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR171a of ＜ 213
The ＞ 46 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCGTTC ACTCAGATCT TACCTGACC
The ＞ 47 of ＜ 210
The ＞ 46 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR171b of ＜ 213
The ＞ 47 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCGGTT CAATCAAATA GTCGTC
The ＞ 48 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR171c of ＜ 213
The ＞ 48 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCGGTG CGGTTCAATC AGA
The ＞ 49 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR172a of ＜ 213
The ＞ 49 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCATGG ACGGTGGTGA TTC
The ＞ 50 of ＜ 210
The ＞ 47 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR172b of ＜ 213
The ＞ 50 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCACAT GGAAATTGAT AAATACC
The ＞ 51 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR172d of ＜ 213
The ＞ 51 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCGGGT TTTCTTTTGA GCC
The ＞ 52 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR172e of ＜ 213
The ＞ 52 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCGTTC CCTTTGCTTT CGC
The ＞ 53 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR173 of ＜ 213
The ＞ 53 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTACT TTCGCTTGCA GAG
The ＞ 54 of ＜ 210
The ＞ 47 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR319b of ＜ 213
The ＞ 54 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCAAAT GAATGAATGA TGCGAGA
The ＞ 55 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR390a of ＜ 213
The ＞ 55 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCCGCC ATGATGATCA CAT
The ＞ 56 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR390b of ＜ 213
The ＞ 56 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTGGC TCACCAGTGC TGT
The ＞ 57 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR391 of ＜ 213
The ＞ 57 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCAGTG GTGACGGTAT CTC
The ＞ 58 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR393a of ＜ 213
The ＞ 58 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTTGG CAAATAAATC ACA
The ＞ 59 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR393b of ＜ 213
The ＞ 59 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTCAA TCGAAAGATG GAA
The ＞ 60 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR394a of ＜ 213
The ＞ 60 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCGCAT TCTGTCCACC TCC
The ＞ 61 of ＜ 210
The ＞ 49 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR394b of ＜ 213
The ＞ 61 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCAATA AGTGTACGTA TCTACGGTG
The ＞ 62 of ＜ 210
The ＞ 44 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR396a of ＜ 213
The ＞ 62 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCCTTA CGCATAAAAT AGTG
The ＞ 63 of ＜ 210
The ＞ 46 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR396b of ＜ 213
The ＞ 63 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTCTT AAACAAAAGT AAGAAG
The ＞ 64 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR398a of ＜ 213
The ＞ 64 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCGGAG TGGCATGTGA ACA
The ＞ 65 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR398b of ＜ 213
The ＞ 65 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCACGG CTGTAATGAC GCT
The ＞ 66 of ＜ 210
The ＞ 44 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR398c of ＜ 213
The ＞ 66 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCCGAG CAATCAACGG CTAT
The ＞ 67 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR399c of ＜ 213
The ＞ 67 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCGCAG GCGACTTGGC TAT
The ＞ 68 of ＜ 210
The ＞ 44 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR400 of ＜ 213
The ＞ 68 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTCAC TACATTTGGT AAGC
The ＞ 69 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR403 of ＜ 213
The ＞ 69 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCATTC AACAGGCTTT ATG
The ＞ 70 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR408 of ＜ 213
The ＞ 70 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCCCTC TTCCCTGGCT CCC
The ＞ 71 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR5663 of ＜ 213
The ＞ 71 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCGGAT TTGCATTCTC ATG
The ＞ 72 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR822 of ＜ 213
The ＞ 72 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCAATG CTTTCTACAG GAA
The ＞ 73 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR824 of ＜ 213
The ＞ 73 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTTGG GGAGTGGGGA GAT
The ＞ 74 of ＜ 210
The ＞ 49 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR837 of ＜ 213
The ＞ 74 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTACA CTCATAATCT TGAAACGAA
The ＞ 75 of ＜ 210
The ＞ 43 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR840 of ＜ 213
The ＞ 75 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCAATC CGCACGATGA TCT
The ＞ 76 of ＜ 210
The ＞ 45 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR844 of ＜ 213
The ＞ 76 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCCTTC TACGCATTGG GCTTA
The ＞ 77 of ＜ 210
The ＞ 49 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR846 of ＜ 213
The ＞ 77 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTAGT TTTGAATTGA AGTGCTTGA
The ＞ 78 of ＜ 210
The ＞ 45 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR851 of ＜ 213
The ＞ 78 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCTGAA AACAATCATC ACGAG
The ＞ 79 of ＜ 210
The ＞ 48 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ miR864 of ＜ 213
The ＞ 79 of ＜ 400
GTTCAGAGTT CTACAGTCCG ACGATCAAAT ACCTTGAAAC TATAAACC
The ＞ 80 of ＜ 210
The ＞ 22 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ Anti-sense of ＜ 213
The ＞ 80 of ＜ 400
GCCTTGGCAC CCGAGAATTC CA
The ＞ 81 of ＜ 210
The ＞ 50 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ 2nd PCR Forward primer of ＜ 213
The ＞ 81 of ＜ 400
AATGATACGG CGACCACCGA GATCTACACG TTCAGAGTTC TACAGTCCGA
The ＞ 82 of ＜ 210
The ＞ 63 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ 2nd PCR Anti-sense of ＜ 213
The ＞ 82 of ＜ 400
CAAGCAGAAG ACGGCATACG AGATCGTGAT GTGACTGGAG TTCCTTGGCA CCCGAGAATT 60
CCA 63

Claims (2)

1. the method that one kind builds the RACE-seq of pre-miRNA 3 ' libraries in plant, including it is as follows：
(1), the extraction of plant total serum IgE；
(2), the preparation of RNA：
The plant total serum IgE for being extracted is separated using SDS-PAGE, the RNA between 50bp-400bp is carried out to cut glue time Receive；
Characterized in that,
(3), connect：
The length of the RNA for having reclaimed concentrates on 50bp-400bp, connects a kind of special in the 3 ' ends of RNA by ligase DNA joints, its sequence is as shown in sequence table SEQ ID No.1；
(4), reverse transcription：
Using reverse transcription reagent box, the RNA to having added good joint carries out reverse transcription, and reverse transcription is into cDNA；
(5), nest-type PRC reaction：
Using the special primer at 5 ' ends, the reverse transcription cDNA with step (4) carries out first round PCR as template；PCR primer is run After PAGE glue is reclaimed, recycling second to take turns primer carries out the second wheel PCR, and PCR primer sends to sequencing after reclaiming.
2. the method that one kind according to claim 1 builds the RACE-seq of pre-miRNA 3 ' libraries in plant, its feature Be, step (5) first round PCR, upper primer as shown in sequence table SEQ ID No.2-SEQ ID No.79, lower primer such as sequence Shown in table SEQ ID No.80；
Second wheel PCR, as shown in sequence table SEQ ID No.81, lower primer is as shown in sequence table SEQ ID No.82 for upper primer.
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107267655A (en) * 2017-08-15 2017-10-20 福建医科大学附属口腔医院 The method that dental pulp stem cell and tip of a root dental papilla stem cell difference miRNAs express spectras are analyzed using two generation sequencing technologies
CN107385516A (en) * 2017-07-20 2017-11-24 深圳大学 The method in the RACE seq libraries of pri miRNA 3 ' in one kind structure plant
CN108103173A (en) * 2017-11-10 2018-06-01 中山大学 A kind of method for building mouse miRNA sequencing libraries and carrying out high-flux sequence
CN111635930A (en) * 2020-05-12 2020-09-08 中国农业科学院农业基因组研究所 Method for high-throughput sequencing and calling full-length sequence of unknown RNA
EP3798319A1 (en) 2019-09-30 2021-03-31 Diagenode S.A. An improved diagnostic and/or sequencing method and kit
EP3828283A1 (en) * 2019-11-28 2021-06-02 Diagenode S.A. An improved sequencing method and kit
CN116042770A (en) * 2022-11-01 2023-05-02 苏州京脉生物科技有限公司 Method and kit for preparing miRNA library in urine and quantifying expression

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007044057A2 (en) * 2005-02-24 2007-04-19 The Ohio State University Research Foundation Methods for quantifying microrna precursors
US20120058521A1 (en) * 2008-08-08 2012-03-08 President And Fellows Of Harvard College Enzymatic oligonucleotide pre-adenylation
CN105349533A (en) * 2015-12-21 2016-02-24 生工生物工程（上海）股份有限公司 Method for constructing strand-specific transcriptome library

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007044057A2 (en) * 2005-02-24 2007-04-19 The Ohio State University Research Foundation Methods for quantifying microrna precursors
US20120058521A1 (en) * 2008-08-08 2012-03-08 President And Fellows Of Harvard College Enzymatic oligonucleotide pre-adenylation
CN105349533A (en) * 2015-12-21 2016-02-24 生工生物工程（上海）股份有限公司 Method for constructing strand-specific transcriptome library

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIU XUHANG等: "A MicroRNA Precursor Surveillance System in Quality Control of MicroRNA Synthesis", 《MOLECULAR CELL》 *
张玥: "低温胁迫下茶树microRNA及其靶基因的识别、鉴定、与差异表达分析", 《中国博士学位论文全文数据库 农业科技辑》 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107385516A (en) * 2017-07-20 2017-11-24 深圳大学 The method in the RACE seq libraries of pri miRNA 3 ' in one kind structure plant
CN107267655A (en) * 2017-08-15 2017-10-20 福建医科大学附属口腔医院 The method that dental pulp stem cell and tip of a root dental papilla stem cell difference miRNAs express spectras are analyzed using two generation sequencing technologies
CN108103173A (en) * 2017-11-10 2018-06-01 中山大学 A kind of method for building mouse miRNA sequencing libraries and carrying out high-flux sequence
CN108103173B (en) * 2017-11-10 2021-04-27 中山大学 Method for constructing mouse miRNA sequencing library for high-throughput sequencing
EP3798319A1 (en) 2019-09-30 2021-03-31 Diagenode S.A. An improved diagnostic and/or sequencing method and kit
US11788137B2 (en) 2019-09-30 2023-10-17 Diagenode S.A. Diagnostic and/or sequencing method and kit
EP3828283A1 (en) * 2019-11-28 2021-06-02 Diagenode S.A. An improved sequencing method and kit
CN111635930A (en) * 2020-05-12 2020-09-08 中国农业科学院农业基因组研究所 Method for high-throughput sequencing and calling full-length sequence of unknown RNA
CN111635930B (en) * 2020-05-12 2023-10-24 中国农业科学院农业基因组研究所 Method for extracting unknown RNA full-length sequence by high-throughput sequencing
CN116042770A (en) * 2022-11-01 2023-05-02 苏州京脉生物科技有限公司 Method and kit for preparing miRNA library in urine and quantifying expression
CN116042770B (en) * 2022-11-01 2023-12-01 苏州京脉生物科技有限公司 Method and kit for preparing miRNA library in urine and quantifying expression

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