CN106399510A - Method for analyzing urine exosome miRNA - Google Patents

Method for analyzing urine exosome miRNA Download PDF

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CN106399510A
CN106399510A CN201610849797.1A CN201610849797A CN106399510A CN 106399510 A CN106399510 A CN 106399510A CN 201610849797 A CN201610849797 A CN 201610849797A CN 106399510 A CN106399510 A CN 106399510A
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黄波
王小中
邹叶青
刘静
徐颜美
闵清华
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Second Affiliated Hospital to Nanchang University
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Abstract

The invention provides a method for analyzing urine exosome miRNA, comprising: collecting a urine sample, extracting exosomes and their total RNA, establishing a cDNA library, performing Illumina sequencing, and processing and annotating the sequencing results to obtain expression information of sample miRNA expression quantity; urine sample exosome miRNA is analyzed by high-throughput sequencing technology to acquire expression information of miRNA; a database for IgA nephrotic patient and a healthy control patient is established, differentially expressed miRNA is deeply studied through target gene function prediction, the deep study on the differentially expressed miRNA helps further illustrate the pathogenesis of IgA nephropathy, and novel method and theoretical basis is provided for the diagnosis of IgA nephropathy. The method is reasonable and feasible in design, a noninvasive precise differential expression miRNA method can be effectively established, and novel ideas are provided for clinical diagnosis and treatment.

Description

A kind of analysis method of urine excretion body miRNA
Technical field
The present invention relates to biomedical, more particularly, to a kind of analysis method of urine excretion body miRNA.
Background technology
Urine is conventional specimen in Clinical detection, due to can be obtained by noninvasive operation and easily collection etc. is excellent in a large number Point, and become the important Specimen origin of diagnosis of kidney disease.At present with regard to the research master of kidney disease biomarker in urine Protein science to be concentrated on and metabolism group aspect, however these albumen and metabolite class biomarker be easily subject to other because Element interference, has high demands to Saving specimen.
Excretion body had once been considered as once skimble-skamble cell " dust ", but subsequent research confirms that it is bio information Important carrier.Albumen is included containing many bioactive molecules in excretion body（Somatomedin and cytokine etc.）, mRNA and MicroRNA, becomes disease diagnosis marker for it and provides material base.Researchers carry out the analysis of excretion vivo protein and send out Existing, the albumen containing many cell-specifics from kidney in excretion body, and research confirms excretion in urine further Body is highly stable, has the potential of the biomarker becoming diagnosis of kidney disease.Comprise in urine to come from cell and dissociate The nucleic acid of DNA, these Exogenous Nucleic Acids can not become the stable source of mark because they may be also from thin in apoptosis Born of the same parents, they transcription group change can not response function cell pathologic condition.Research finds, contains and compile in urine excretion body Code nephron and collecting tubule all albumen mRNA it is seen that in urine excretion body contain be possible to react renal function nucleic acid, How to obtain the necessary problem that miRNA in accurate urine excretion body is those skilled in the art's deep structure research.
Content of the invention
For deficiencies of the prior art, the present invention solves existing analysis method and there is complex operation, error relatively Big problem, provides a kind of analysis method of urine excretion body miRNA, provides necessary data ginseng for later phase clinical medical research Examine.
Solve above-mentioned technical problem, the present invention adopts the following technical scheme that：A kind of analysis side of urine excretion body miRNA Method is it is characterised in that comprise the following steps：
（1）The collection of urine specimen：
The urina sanguinis 120ml collecting individuals is as urine specimen；
（2）Excretion body and its extraction of total serum IgE：
The urine specimen collected is pressed following centrifugation step and extracts excretion body：First press 400g, 2000g and 10000g centrifugal force successively It is centrifuged 10min, 20min and 30min respectively, to remove the bulky grains such as the cell in urine specimen and cell debriss, collect supernatant Liquid；Then the supernatant utilizing Ultracentrifuge to collect before 200000g centrifugal force, the time is 1 hour, abandons supernatant Obtain gelatinous precipitate；Then use the resuspended precipitation of PBS, 200000g is centrifuged 1 hour again；Collect precipitation, add Trizol examination Agent, -80 DEG C preserve in case RNA extracts；
Extract sample total serum IgE：Defrosting sample under room temperature, adds 0.2ml/1mlTrizol chloroform, acutely shakes 15s, and room temperature is placed 2-3min, 4 DEG C of 12000g are centrifuged 15min；Draw upper strata aqueous phase, add 0.5ml/1mlTrizol isopropanol, room temperature is placed 10min, 4 DEG C of 12000g are centrifuged 10min；Remove supernatant, with 1ml 75% washing with alcohol RNA precipitate, 4 DEG C of 7500g centrifugation 5min； Go to precipitate, 8min is dried, add RNA lysate, -80 DEG C save backup；
Using Agilent 2200 TapeStation and ND-1000 Nanodrop instrument, concentration is carried out to the RNA sample extracted And quality testing；
（3）The foundation of cDNA library and Illumina sequencing：
With 1 μ g initial amount, carry out library with the supporting TruSeqR Small RNA Sample Prep Kit of Illumina company Build：First RNA is carried out with 3 ' ends and 5 ' ends connect modification, then utilize reverse transcription reagents to synthesize the first chain cDNA, expanded using PCR Increase and obtain DNA product, after isolate small molecule DNA through polyacrylamide gel electrophoresis, final obtain high-flux sequence DNA literary composition Storehouse, and with Agilent 2200 TapeStation, quality inspection is carried out to library；
The library of preparation is carried out upper machine sequencing according to Illumina Hiseq 2500 sequenator operating guidance, higher level's sample Final concentration of 10pM；
（4）The process of sequencing result and annotation
Illumina Hiseq 2500 is sequenced the sequence sets of gained, by removing joint, removing low quality sequence and depolluting and waited Journey completes the preliminary filtration of data, obtains clean sequence；By the clean sequence and the miRBase that obtain, Genebank, UCSC, NONCODE and Rfam data base compares, and obtains tiny RNA classification and the annotation result of two groups of samples；By all small RNA fragments After annotation, carry out new miRNA prediction with the remaining fragment that do not annotate；
（5）Sample miRNA expression
By the sequencing data of gained by the biomolecule information database with miRNA（miRBase20.0）It is compared, obtain The expressing information of miRNA.
Compared to existing technology, the invention has the advantages that：
1st, the present invention passes through high throughput sequencing technologies, and urine specimen excretion body miRNA is analyzed and processed, and obtains the expression of miRNA Information；By setting up the data base of IgA nephropathy patient and normal healthy controls person, obtain the difference of two kinds of urine specimen excretion body miRNA Different expression, is predicted the miRNA of further investigation differential expression, furthers investigate these differential expressions using target gene function MiRNA will be helpful to be further elucidated with the morbidity of IgA nephropathy and pathogenesis.For IgA nephropathy diagnosis provide new method and Theoretical basiss.
2nd, the inventive method is reasonable in design feasible, can effectively set up a kind of noninvasive accurately differential expression MiRNA method, provides new thinking for clinical diagnosis and treatment work.
3rd, the method is easy to operate, and detection data is reliable.
4th, establish a kind of analysis method of new nephropathy, the method adopts urine specimen analysis excretion body miRNA expression Situation, is a kind of noninvasive analysis method.The data obtained is directly compared with data base, can quickly obtain analysis knot Really.
Brief description
Fig. 1 is classification and the annotated map of the clean small RNA fragments of IgA nephropathy group high-flux sequence gained；
Fig. 2 is classification and the annotated map of the clean small RNA fragments of healthy control group high-flux sequence gained；
Fig. 3 IgA nephropathy group and the urine excretion body miRNA differential expression cluster analyses figure of healthy control group.
Specific embodiment
Below by way of specific embodiment and combine accompanying drawing, technical scheme is described in further detail.Remove Specialize, the routine that technological means used in following examples and operational approach are well known to those skilled in the art Means and method, institute is commercial goods using raw material.
1st, the collection of urine specimen
The present embodiment is chosen 3 clinics respectively and has been diagnosed as IgA nephropathy patient and agematched physical examination of healthy population, receives respectively The urina sanguinis of collection 120ml, is used for subsequent experimental as IgA nephropathy group and healthy control group and operates.6 specimen are all from University Of Nanchang Second Affiliated Hospital, clinic is made a definite diagnosis, and exclusion has the patient that other can cause renal glomerular disease and complication, all IgA kidneys Sick urine specimen is all collected before treatment and medication.
2nd, the extraction of excretion body and its total serum IgE
The urine specimen collected is pressed following centrifugation step and extracts excretion body：First press 400g, 2000g and 10000g centrifugal force successively It is centrifuged 10min, 20min and 30min respectively, to remove the bulky grains such as the cell in urine specimen and cell debriss, collect supernatant Liquid；Then the supernatant utilizing Ultracentrifuge to collect before 200000g centrifugal force, the time is 1 hour, abandons supernatant Obtain gelatinous precipitate；Finally use the resuspended precipitation of PBS, 200000g is centrifuged 1 hour again；Collect precipitation, add Trizol examination Agent, -80 DEG C preserve in case RNA extracts.
Respectively by obtain by above-mentioned steps two groups of experimental group samples with organizing mixed in equal amounts, extract sample total serum IgE, step is such as Under：Defrosting sample under room temperature, adds 0.2ml/1mlTrizol chloroform, acutely concussion 15s, room temperature placement 2-3min, 4 DEG C 12000g is centrifuged 15min；Absorption upper strata aqueous phase, addition 0.5ml/1mlTrizol isopropanol, room temperature placement 10min, 4 DEG C 12000g is centrifuged 10min；Remove supernatant, with 1ml 75% washing with alcohol RNA precipitate, 4 DEG C of 7500g centrifugation 5min；Go to precipitate, be dried 8min, adds RNA lysate, -80 DEG C save backup.
Using Agilent 2200 TapeStation and ND-1000 Nanodrop instrument, the RNA sample extracted is carried out Concentration and quality testing.
3rd, the foundation of cDNA library and Illumina sequencing
The sample that above-mentioned quality inspection is passed through, with 1 μ g initial amount, with the TruSeqR Small RNA that Illumina company is supporting Sample Prep Kit carries out library construction, and step is as follows：First RNA is carried out with 3 ' ends and 5 ' ends connect modification, then utilize inverse Transcript reagent synthesize the first chain cDNA, using PCR amplification obtain DNA product, after isolate through polyacrylamide gel electrophoresis Small molecule DNA, final acquisition high-flux sequence DNA library, and with Agilent 2200 TapeStation, matter is carried out to library Inspection.
The library of preparation is carried out upper machine sequencing, higher level's sample according to Illumina Hiseq 2500 sequenator operating guidance This final concentration of 10pM.The preparation of DNA library and high-flux sequence are all in the assistance of Guangzhou Rui Bo bio tech ltd Under complete.
4th, the process of sequencing result and annotation
Illumina Hiseq 2500 is sequenced the sequence sets of gained, by removing joint, removing low quality sequence and depolluting and waited Journey completes the preliminary filtration of data, obtains clean sequence；By the clean sequence and the miRBase that obtain, Genebank, UCSC, NONCODE and Rfam data base compares, and obtains tiny RNA classification and the annotation result of two groups of samples（Referring to Fig. 1, Fig. 2）；Will After all small RNA fragments annotations, carry out new miRNA prediction with the remaining fragment that do not annotate.
5th, the analysis of two groups of sample miRNA expressions
By the sequencing data of gained by the biomolecule information database with miRNA（miRBase20.0）It is compared, obtain each group The expressing information of miRNA.Analyze two groups of marks using Cufflink software and Benjimini and Hochberg statistical method MiRNA differential expression situation in this, p≤0.01 and log2 (fold change) >=1.5 represent that difference is statistically significant.
6th, two groups of inter-sample difference express miRNA result
By above-mentioned analysis, result shows, has the miRNA of 158 differential expressions, wherein between IgA nephropathy group and healthy control group 21 miRNA are high significant difference, and 20 miRNA are general significant difference（log2（Fold Change）Absolute value is more than 1, P< 0.01 is high significant difference；log2（Fold Change）Absolute value is more than 1, P<0.05 is general significant difference）.As table 1 institute Show.Nephropathy group（A）With healthy control group（B）The miRNA expression cluster situation of 158 significant difference expression is referring to Fig. 3.
Table 1 IgA nephropathy group（IgAN）With Normal group（HC）The urine excretion body miRNA of differential expression
miRNA_ID HC expression values IgAN expression values log2(foldchange) Significance-Lable
hsa-miR-27a-3p 6029.0324 12.6323 8.8987 **
hsa-miR-31-5p 221.9788 55.0982 2.0104 **
hsa-miR-10b-3p 2.5073 66.3866 4.7267 **
hsa-miR-34a-5p 19.3897 1.6126 3.5878 **
hsa-miR-181c-5p 12.5365 0 16.9358 **
hsa-miR-211-5p 0 18.9484 17.5317 **
hsa-miR-215-5p 0 6792.803 26.0175 **
hsa-let-7g-5p 125.6989 38.4344 1.7095 **
hsa-miR-30b-5p 58.3363 14.3793 2.0204 **
hsa-miR-126-3p 2.3401 161.5318 6.1091 **
hsa-miR-30e-3p 8.1905 3759.7137 8.8425 **
hsa-miR-365b-3p 21.897 0 17.7404 **
hsa-miR-135b-5p 172.8359 0 20.721 **
hsa-miR-451a 0 30.3712 18.2123 **
hsa-miR-486-5p 0 110.1964 20.0716 **
hsa-miR-146b-3p 0 26.3396 18.0069 **
hsa-miR-509-3p 0 16.9326 17.3694 **
hsa-miR-514a-3p 0 14.7824 17.1735 **
hsa-miR-708-3p 0 94.6077 19.8516 **
hsa-miR-374b-5p 174.8418 56.4421 1.6312 **
hsa-miR-378i 0 7328.3308 26.127 **
hsa-miR-16-5p 116.3383 54.0231 1.1067 *
hsa-miR-29a-3p 74.0487 32.9245 1.1693 *
hsa-miR-10a-3p 21.2284 6.9881 1.603 *
hsa-miR-205-5p 142.247 58.3235 1.2863 *
hsa-miR-125a-5p 920.1759 412.5646 1.1573 *
hsa-miR-146a-5p 59.3392 410.6832 2.791 *
hsa-miR-186-5p 153.2791 67.0586 1.1927 *
hsa-miR-29c-5p 9.0262 1.747 2.3692 *
hsa-miR-29c-3p 424.0665 206.5511 1.0377 *
hsa-miR-361-3p 31.5919 224.1556 2.8269 *
hsa-miR-365a-3p 6.6861 0 16.0289 *
hsa-miR-374a-5p 235.8525 102.2677 1.2055 *
hsa-miR-378a-3p 770.0727 5505.7895 2.8378 *
hsa-miR-326 6.6861 0 16.0289 *
hsa-miR-508-3p 0 9.6758 16.5621 *
hsa-miR-151b 103.1332 44.6161 1.2089 *
hsa-miR-320c 0 13.4386 17.036 *
hsa-miR-378d 0 9.407 16.5214 *
hsa-miR-548o-3p 0 11.2884 16.7845 *
hsa-miR-500b-5p 18.2196 5.3754 1.761 *
Note：Significance-Lable represents significance labelling, if | log2 (Fold Change) |>1 and P-value< 0.01, then Significance-Lable is " * * "；If | log2 (Fold Change) |>1 and P-value<0.05, then Significance-Lable is " * ".
To sum up, the present invention passes through high throughput sequencing technologies, and urine specimen excretion body miRNA is analyzed and processed, and obtains miRNA Expressing information；By setting up the data base of IgA nephropathy patient and normal healthy controls person, obtain two kinds of urine specimen excretion bodies The differential expression situation of miRNA, the miRNA furtheing investigate these differential expressions will be helpful to be further elucidated with sending out of IgA nephropathy Disease and pathogenesis.The method is easy to operate, and detection data is reliable.There is provided necessary data refer for clinic study.
It should be pointed out that the above embodiment can make those skilled in the art that the answering of the present invention is more fully understood With, but limit the present invention never in any form.Therefore, although this specification to present invention has been detailed description, It will be appreciated by those skilled in the art that still the present invention can be modified or equivalent；And all are without departing from this The technical scheme of bright spirit and its improvement, it all should be covered in the middle of the protection domain of patent of the present invention.

Claims (2)

1. a kind of analysis method of urine excretion body miRNA is it is characterised in that comprise the following steps：
（1）The collection of urine specimen：
The urina sanguinis 120ml collecting individuals is as urine specimen；
（2）Excretion body and its extraction of total serum IgE：
The urine specimen collected is pressed following centrifugation step and extracts excretion body：First press 400g, 2000g and 10000g centrifugal force successively It is centrifuged 10min, 20min and 30min respectively, to remove the bulky grains such as the cell in urine specimen and cell debriss, collect supernatant Liquid；Then the supernatant utilizing Ultracentrifuge to collect before 200000g centrifugal force, the time is 1 hour, abandons supernatant Obtain gelatinous precipitate；Then use the resuspended precipitation of PBS, 200000g is centrifuged 1 hour again；Collect precipitation, add Trizol examination Agent, -80 DEG C preserve in case RNA extracts；
Extract sample total serum IgE：Defrosting sample under room temperature, adds 0.2ml/1mlTrizol chloroform, acutely shakes 15s, and room temperature is placed 2-3min, 4 DEG C of 12000g are centrifuged 15min；Draw upper strata aqueous phase, add 0.5ml/1mlTrizol isopropanol, room temperature is placed 10min, 4 DEG C of 12000g are centrifuged 10min；Remove supernatant, with 1ml 75% washing with alcohol RNA precipitate, 4 DEG C of 7500g centrifugation 5min； Go to precipitate, 8min is dried, add RNA lysate, -80 DEG C save backup；
Using Agilent 2200 TapeStation and ND-1000 Nanodrop instrument, concentration is carried out to the RNA sample extracted And quality testing；
（3）The foundation of cDNA library and Illumina sequencing：
With 1 μ g initial amount, carry out library with the supporting TruSeqR Small RNA Sample Prep Kit of Illumina company Build：First RNA is carried out with 3 ' ends and 5 ' ends connect modification, then utilize reverse transcription reagents to synthesize the first chain cDNA, expanded using PCR Increase and obtain DNA product, after isolate small molecule DNA through polyacrylamide gel electrophoresis, final obtain high-flux sequence DNA literary composition Storehouse, and with Agilent 2200 TapeStation, quality inspection is carried out to library；
The library of preparation is carried out upper machine sequencing according to Illumina Hiseq 2500 sequenator operating guidance, higher level's sample Final concentration of 10pM；
（4）The process of sequencing result and annotation
Illumina Hiseq 2500 is sequenced the sequence sets of gained, by removing joint, removing low quality sequence and depolluting and waited Journey completes the preliminary filtration of data, obtains clean sequence；By the clean sequence and the miRBase that obtain, Genebank, UCSC, NONCODE and Rfam data base compares, and obtains tiny RNA classification and the annotation result of two groups of samples；By all small RNA fragments After annotation, carry out new miRNA prediction with the remaining fragment that do not annotate；
（5）Sample miRNA expression
By the sequencing data of gained by the biomolecule information database with miRNA（miRBase20.0）It is compared, obtain The expressing information of miRNA.
2. urine excretion body miRNA as claimed in claim 1 analysis method it is characterised in that：Step（5）In specific practice It is to remove repetitive sequence and the mRNA degradation fragment being more than 13nt with known array position intersection；Using SOAP2.0 software The clean small RNA molecular sequence obtaining and RNA sequence in Genebank, UCSC, NONCODE and Rfam data base are compared To analysis, remove rRNA, tRNA, snRNA, snoRNA and piRNA；Again by remaining sequence and people miRNA in miRNA data base Sequence compares.
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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
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CN106939306A (en) * 2017-04-11 2017-07-11 江苏吉诺思美精准医学科技有限公司 It is a kind of based on PSP suitable for high-flux sequence elRNA library constructing methods
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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
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