CN103334159A - Method of constructing degradome sequencing library - Google Patents

Abstract

The invention discloses a method of constructing degradome sequencing library. The method comprises following steps: sorting mRNA sections of miRNA target gene, connecting connectors of 5' terminals of mRNA sections of miRNA target gene, and amplifying cDNA of miRAN target gene. The technical scheme facilitates the operation, enables the specific loci where the animal and plant miRNAs act on the target gene to be located accurately, breaks through the bioinformatics prediction limitation, finds the target gene where miRNA acts on, and has the characteristics of efficiency, rapidness, intuition and accuracy.

Description

A kind of method that makes up degraded group order-checking library

Technical field

The present invention relates to biological technical field, more specifically relate to a kind of method that makes up degraded group order-checking library

Background technology

MiRNA(microRNA) be the endogenous non-coding RNA that a class length is about 22 Nucleotide, almost participated in all important vital movements in the animal and plant body, the regulation and control Expression of Related Genes.Usually, miRNA is by partially or completely matching with target gene (mRNA), the translation inhibition or the restriction endonuclease of gene that cause gene shear to realize that to the expression of gene regulation and control translation with gene suppresses in the majority in animal body, then are sheared with target gene to occupy the majority in plant materials.Utilize ripe miRNA micro-array chip or qPCR technology can efficiently filter out the miRNA of differential expression in the animal and plant body rapidly, and the target gene of finding out difference miRNA effect become the key of its important biomolecule function of research.

Be different from animal, thereby Mirnas of plant is common and target gene carries out fully or approach the expression that pairing completely causes the shearing regulatory gene of target gene.Initial scientific research personnel utilizes this feature that the target gene of Mirnas of plant has been launched the information biology prediction, and has obtained many significant achievements in research.Yet because Forecasting Methodology can't be distinguished the true and false of prediction target gene, all predict the outcome must be through experiment confirm to eliminate the false and retain the true.This has greatly influenced the exploration efficient of target gene.Simultaneously in some cases, when having higher mispairing between miRNA and target gene, Forecasting Methodology may be lost many real target genes.

Follow appearance and the development of order-checking of future generation (Next Generation Sequencing) or title high-flux sequence (High-throughput Sequencing), recently a kind of new experimental technique occurs and be called degraded group order-checking (Degradome Sequencing), it combines high throughput sequencing technologies and bioinformatic analysis advantage separately, be successfully applied to Arabidopis thaliana 1,2, the miRNA target gene screening of paddy rice 3 plants such as grade.The principle of degraded group order-checking is that most miRNA utilizes shearing action regulation and control target gene expression in plant materials, and shearing often occurs on the tenth Nucleotide of miRNA and mRNA complementary region.Target gene produces two fragments through shearing, and 5 ' shears fragment and 3 ' shears fragment.Wherein 3 ' shear fragment, include 5 ' monophosphate and 3 ' polyA tail freely, can be connected product and can be used for the downstream high-flux sequence by the RNA ligase enzyme; And contain the complete genome of 5 ' cap sequence, contain that 5 ' of cap sequence is sheared fragment or other RNA that lacks 5 ' monophosphate group can't be connected by the RNA enzyme, thereby can't enter the order-checking experiment in downstream; Sequencing data is carried out compare of analysis in depth, can find intuitively a crest can occur in certain site of mRNA sequence, and this locates candidate's miRNA shearing site just.Utilize the order-checking of degraded group, the scientific research personnel has broken away from the restriction of information biology prediction, has really found the effect target gene of miRNA from experiment.

Summary of the invention

The object of the invention has been to provide a kind of construction process of the group order-checking library of degrading, and this method can accurately find animals and plants miRNA to act on the concrete site of target gene, simple to operation and visual result.

In order to achieve the above object, the present invention is by the following technical solutions: a kind of method that makes up degraded group order-checking library may further comprise the steps:

1) sorting of .miRNA target gene mRNA fragment: with 3 ' joint (3 ' Adapter sequence: 5 '-TGGAA TTCTC GGGTG CCAAG G-3 ') complementary with 3 ' end annealing of target gene mRNA fragment, its reaction conditions is:

Above-mentioned connection mixture in 65 ℃ of insulations 3 minutes, is obtained to contain the connection mixture that 3 ' joint-mRNA connects product;

2). according to the reverse transcription of right 1 described connection product: the 3 ' joint-mRNA in the connection mixture that step 1 is obtained connects product and connects 5 ' joint (5 ' Adapter sequence: 5 '-GUUCA GAGUU CUACA GUCCG ACGAU C-3 '), reverse transcription obtains the target gene mRNA fragment that two ends all have joint, the steps include:

A) 5 ' of .mRNA joint connects:

Above-mentioned reaction system in 37 ℃ of insulations 1-2 hour, is placed on ice afterwards;

B). purifying:

In reaction system, add 75 μ l H ₂The O dehydrated alcohol was placed 30 minutes for-80 ℃, and centrifugal 30 minutes of 16000g removes the supernatant postlyophilization, adds 19 μ l RNase Free H ₂O; Obtain the connection product of purifying;

C). reverse transcription:

Said mixture was left standstill 2 minutes in 25 ℃, add 1 μ l ThermoScript II, in 25 ℃ of insulations 10 minutes, again in 42 ℃ of insulations 40 minutes, at last in 70 ℃ of insulations 20 minutes;

3) amplification of .miRNA target gene cDNA: the target gene mRNA fragment that all has joint with two ends is template, utilize primer (5 '-AATGA TACGG CGACC ACCGA GATCT ACACG TTCAG AGTTC TACAG TCCGA-3 ' and 5 '-CAAGC AGAAG ACGGC ATACG AGATC GTGAT GTGAC TGGAG TTCCT TGGCA CCCGA GAATT CCA-3 ') to carry out pcr amplification, obtain the degraded group order-checking library of target gene mRNA fragment, the steps include:

1. carry out the PCR reaction:

98 ℃ of sex change 45 seconds, 10-15 PCR circulation, 98 ℃ of sex change 15 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, and 72 ℃ of follow-up extensions 1 minute are stored in 4 ℃ at last;

2. gel electrophoresis is reclaimed: utilize gel electrophoresis to separate the PCR product, cut and expect that sized molecules and recovery obtain high-flux sequence and organize the library with degraded.

The technical solution of the present invention concrete site that can accurately find animals and plants miRNA to act on target gene simple to operate, broken away from the restriction of information biology prediction, really from experiment, find the effect target gene of miRNA, had efficient, quick, intuitive and accurate characteristics.

Description of drawings

Fig. 1 is PCR product amplification, and 1,2,3 is three repetitions of sample, and M is 50bp DNA Marker.Fig. 2 is for cutting expection sized molecules band, and 1,2,3 is three repetitions of sample, and M is 50bp DNA Marker.

Embodiment

Below further set forth technical solution of the present invention with the example that is configured in soybean degraded group library:

1 material and reagent

3 ' joint, 5 ' joint and amplimer are given birth to worker company by Shanghai and are synthesized; MRNA extracts test kit, ligase enzyme and PCR test kit available from Thermo Fisher.

2 methods and step

2.1 the extraction of the fresh and tender blade mRNA of soybean

2.1.1 prepare 65 ℃ and 80 ℃ of water-baths;

2.1.2 100ug total RNA is diluted to 100ul with NF-H2O;

2.1.3 behind 65 ℃ of water-bath 5min, place on ice;

2.1.4 get 2 1.5ml spiral tubes, be respectively charged into 50ul sera-mag oligo (dT) beads;

2.1.5 wash beads twice with 100ul Bead Binding buffer;

2.1.6 with the resuspended beads of 100ul Bead Binding buffer, add 100ul total RNA;

2.1.7 flick 5min, place on the magnetic force frame and place 30s, remove supernatant;

2.1.8 with the magnetic bead in the 200ul washing buffer cleaning step 7, twice;

2.1.9 add 50ul10mM Tris-HCL, 80 ℃, 2min is placed on rapidly on the magnetic force frame;

2.1.10 shift supernatant (mRNA) to new 1.5ml PE pipe, add 50ul Binding buffer therein, 65 ℃, 5min places on ice;

2.1.11 with the magnetic bead in the 200ul washing buffer cleaning spiral tube, twice;

2.1.12 the 100ul mRNA of step 10 is joined in the magnetic bead of the washed spiral tube of step 11, and room temperature is flicked 5min, places 30s on the magnetic force frame, removes supernatant;

2.1.13 clean magnetic bead, twice with 200ul washing buffer;

2.1.14 with the magnetic bead in the 150ul10mM Tris-HCL suspension pipe, 80 ℃, 2min;

2.1.15 rapidly pipe is put on the magnetic force frame, shifts supernatant (mRNA) and manage to 200ul PCR;

2.1.16 with the magnetic bead in the 100ul10mM Tris-HCL suspension pipe, 80 ℃, 2min;

2.1.17 rapidly pipe is put on the magnetic force frame, shifts supernatant (mRNA) to 200ul PCR pipe, cumulative volume 250ul;

2.1.18 survey the OD value;

2.1.19 result: OD260/280=2.18, OD260/230=2.24, concentration 2.05 μ g/ μ L

The sorting of miRNA target gene mRNA fragment

2.2 with 3 ' joint (3 ' Adapter, sequence: 5 '-TGGAA TTCTC GGGTG CCAAG G-3 ') complementary with 3 ' end annealing of target gene mRNA fragment, its reaction conditions is:

Above-mentioned connection mixture is incubated 3 minutes in 65 ℃.

2.3miRNA the connection of target gene mRNA fragment 5 ' end connector

On the basis of said mixture, again mRNA is connected product and connect 5 ' joint (5 ' Adapter:5 '-GUUCA GAGUU CUACA GUCCG ACGAU C-3 '), reverse transcription obtains two ends and all has the target gene mRNA fragment of joint and obtain miRNA target gene cDNA, and its concrete steps are:

2.3.1 the 5 ' joint of fragmentation RNA connects:

Above-mentioned reaction system in 37 ℃ of insulations 1-2 hour, is placed on ice afterwards.

2.3.2 purifying:

Add 75 μ l H2O dehydrated alcohols in reaction system, placed 30 minutes for-80 ℃, centrifugal 30 minutes of 16000g removes the supernatant postlyophilization, adds 19 μ l Rnase Free H2O.

2.3.3 reverse transcription:

Said mixture is left standstill 2min in 25 ℃, add 1 l ThermoScript II, in 25 ℃ of insulations 10 minutes, again in 42 ℃ of insulations 40 minutes, at last in 70 ℃ of insulations 20 minutes.

2.4miRNA the amplification of target gene cDNA

The target gene mRNA fragment that all has joint with two ends is template, utilize primer (forward direction primer, sequence: 5 '-AATGA TACGG CGACC ACCGA GATCT ACACG TTCAG AGTTC TACAG TCCGA-3 ' and reverse primer, sequence: 5 '-CAAGC AGAAG ACGGC ATACG AGATC GTGAT GTGAC TGGAG TTCCT TGGCA CCCGA GAATT CCA-3 ') carry out pcr amplification, obtain target gene mRNA cDNA fragment library, the steps include:

2.4.1 carry out the PCR reaction:

98 ℃ of sex change 45 seconds, 10-15 PCR circulation (if parent material is less, should suitably increase cycle number), 98 ℃ of sex change 15 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, and 72 ℃ of follow-up extension 1min are stored in 4 ℃ at last.

The PCR product is through the 6%TBE gel electrophoresis analysis, and the result as shown in Figure 1

2.4.2 gel electrophoresis is reclaimed

Utilize gel electrophoresis to separate the PCR product, cut and expect that sized molecules and recovery obtain high-flux sequence and organize the library with degraded, as shown in Figure 2.

Successfully obtained the soybean leaves degraded group library of expecting by the disclosure inventive method, PCR product electrophoretogram conforms to expection, reclaims the target molecule that obtains and successfully is used for high-flux sequence.

Claims (1)

1. a method that makes up degraded group order-checking library is characterized in that, may further comprise the steps:

1) sorting of .miRNA target gene mRNA fragment: with 3 ' joint (3 ' Adapter sequence: 5 '-TGGAA TTCTC GGGTG CCAAG G-3 ') complementary with 3 ' end annealing of target gene mRNA fragment, its reaction conditions is:

Above-mentioned connection mixture in 65 ℃ of insulations 3 minutes, is obtained to contain the connection mixture that 3 ' joint-mRNA connects product;

2). 3 ' joint in the connection mixture that step 1 is obtained-mRNA connects product and connects 5 ' joint (5 ' Adapter sequence: 5 '-GUUCA GAGUU CUACA GUCCG ACGAU C-3 '), reverse transcription obtains the target gene mRNA fragment that two ends all have joint, the steps include:

A) 5 ' of .mRNA joint connects:

Above-mentioned reaction system in 37 ℃ of insulations 1-2 hour, is placed on ice afterwards;

B). purifying:

In reaction system, add 75 μ l H ₂The O dehydrated alcohol was placed 30 minutes for-80 ℃, and centrifugal 30 minutes of 16000g removes the supernatant postlyophilization, adds 19 μ l RNase Free H ₂O; Obtain the connection product of purifying;

C). reverse transcription:

Said mixture was left standstill 2 minutes in 25 ℃, add 1 μ l ThermoScript II, in 25 ℃ of insulations 10 minutes, again in 42 ℃ of insulations 40 minutes, at last in 70 ℃ of insulations 20 minutes;

3) amplification of .miRNA target gene cDNA: the target gene mRNA fragment that all has joint with two ends is template, utilize primer (5 '-AATGA TACGG CGACC ACCGA GATCT ACACG TTCAG AGTTC TACAG TCCGA-3 ' and 5 '-CAAGC AGAAG ACGGC ATACG AGATC GTGAT GTGAC TGGAG TTCCT TGGCA CCCGA GAATT CCA-3 ') to carry out pcr amplification, obtain the degraded group order-checking library of target gene mRNA fragment, the steps include:

1. carry out the PCR reaction:

98 ℃ of sex change 45 seconds, 10-15 PCR circulation, 98 ℃ of sex change 15 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, and 72 ℃ of follow-up extensions 1 minute are stored in 4 ℃ at last;

2. gel electrophoresis is reclaimed: utilize gel electrophoresis to separate the PCR product, cut and expect that sized molecules and recovery obtain high-flux sequence and organize the library with degraded.

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