CN108085386B - The identification of the reference gene of osteosarcoma miRNA detection - Google Patents

The identification of the reference gene of osteosarcoma miRNA detection Download PDF

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CN108085386B
CN108085386B CN201710238142.5A CN201710238142A CN108085386B CN 108085386 B CN108085386 B CN 108085386B CN 201710238142 A CN201710238142 A CN 201710238142A CN 108085386 B CN108085386 B CN 108085386B
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朱伟
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Abstract

The invention discloses the internal reference miR-96 gene for stablizing expression in osteosarcoma patient, the combination constituted including hsa-miR-128a-5p or hsa-miR-128a-5p and let-7d.The reference gene can be used for the standardization of target gene in osteosarcoma patient miRNA detection, and the comparison of result of study between different experiments room may be implemented, promote the normalized of each experimental result.

Description

The identification of the reference gene of osteosarcoma miRNA detection
Technical field
The invention patent relates to the identifications to a kind of internal reference miR-96 gene for osteosarcoma miRNA detection.
Background technique
Osteosarcoma (osteosarcoma, OSA) is common malignant bone tumor, is directly produced by the sarcoma cell of malignant proliferation Raw neoplastic osteoid or immature bone, histological characteristic be hyperplasia shuttle shape tumour cell directly generate osteoid matrix or Immature bone, nearly all bone and flesh tumor metastasis are transferred to lung through blood, and minority is transferred to the internal organs such as brain, kidney and through lymph It carries down shifting.The invasion and transfer of osteosarcoma seriously affect the quality of life and prognosis of patient.Although clinical orthopaedics worker is always It is dedicated to the research prevented and treated it, but the overall 5 years survival rates of osteosarcoma still fluctuated in recent years, therefore, searched out bone and flesh Tumor early stage Precise Diagnosis and the Effective target site for the treatment of shoulder heavy responsibilities.
MiRNA is a kind of small molecule non-coding RNA (non-coding being made of 19~22 mature nucleotide RNAmolecules, ncRNAs), it was found for the first time by Ambros seminar when studying nematode in 1993.So far, MiRNA exists in all animals and plants, accounts for about the 4% of genome.They rise in the generation of cell, differentiation, proliferation and apoptosis Important role.In addition, miRNA has extensive adjustment effect to cancer based on the basis of target gene function, they It can be used as tumor suppressor, can also be used as the tumorigenesis factor.Main mechanism of action includes: the missing in the site miRNA, expands Increase, mutation, epigenetic level changes, and turns the unconventionality expression etc. of clever regulatory factor.Expression table of the miRNA in tissue and cell It is now significant cancer-related, tissue specificity and expression stability.And expression of the miRNA in peripheral blood equally has Cancer-related and tissue specificity, while compared with RNA, expression stability is more significant.Existing research passes through height at present Flux sequencing or biochip technology detect the expression of miRNA to determine the relevant miRNA with osteosarcoma, including miR- 4714-3p, miR-1299, miR-6779-5p, miR-1292, miR-3688-3p, miR-5010 etc..
There is various factors to can lead to sample variation, such as the quality and quantity of RNA in gene expression research, but expresses Data can correct this deviation by the correction of reference gene.The selection of miRNA reference gene is extremely important.There is research table Bright, the selection of reference gene can make a significant impact RT-PCR result, if reference gene selection is inappropriate, will make between sample Gene expression difference is blanked or excessively exaggerates, and leads to mistake conclusion even opposite with the fact occur.Further, since The specific expressed and tumor type of miRNA is closely related, therefore need to consider that tumor type, sample come when selecting reference gene The factors such as source and experimental method, pointedly to select suitable reference gene.Occur manually synthesizing in recent years non- Exogenous control of the mankind (such as nematode) miRNA as miRNA standardized testing, still, exogenous control cannot correct sample The difference of acquisition, so being not preferably to select.Meanwhile some endogenous genes are often used as tissue/cell miRNA detection Reference gene, such as 5SrRNA, 18SrRNA and U6 etc., but since these genes are not miRNA, cannot represent miRNA's Component, and the efficiency of the extractions of these genes, reverse transcription and PCR amplification may be different with miRNA.Therefore, these bases Because nor optimal selection.Research best reference gene standardized to miRNA in osteosarcoma research there is no to carry out at present Systematic identification and assessment therefore can be as microRNA standardization in osteosarcoma research there is an urgent need in the art to develop Reference gene, and establish detection miRNA, especially recycle miRNA effective standard scheme.
Summary of the invention
To solve the above problems, the invention discloses a kind of identifications of the internal reference miR-96 gene of osteosarcoma miRNA detection.
Specifically, the present invention provides a kind of application of miRNA in preparation for the internal reference reagent of osteosarcoma miRNA detection, It is characterized by: the miRNA is hsa-miR-128a-5p.
Application the present invention also provides the combination of miRNA a kind of in preparation for the internal reference reagent of osteosarcoma miRNA detection, It is characterized by: described group is combined into the combination that hsa-miR-128a-5p and let-7d is constituted.
The wherein sequence of hsa-miR-128a-5p are as follows: the sequence of 5 '-CGGGGCCGUAGCACUGUCUGAGA-3 ', let-7d It is classified as: 5 '-AGAGGUAGUAGGUUGCAUAGUU-3 '.
The present invention also provides the internal reference reagents detected for osteosarcoma miRNA in the kit of preparation diagnosis osteosarcoma Using.A kind of kit for diagnosing osteosarcoma is provided, which contains for detecting internal reference miRNA hsa-miR-128a-5p Or the combined reagent of hsa-miR-128a-5p and let-7d.
Further, the kit also includes for RNA separating liquid, examination needed for miRNA extraction and qRT-PCR reaction Agent and enzyme and standard items or/and reference substance.
The present invention also provides a kind of method of the relative expression quantity of miRNA in detection Patients with Osteosarcoma blood plasma or serum, packets Include following steps:
(1) it extracts the total serum IgE of test plasma or serum and reverse transcription is cDNA；
(2) using step (1) obtain cDNA as template, detect respectively wherein the content of the coded sequence of purpose miRNA with The content of the coded sequence of hsa-miR-128a-5p；
(3) calculate purpose miRNA's as a result, using the hsa-miR-128a-5p as reference gene according to step (2) Relative expression quantity.
Further, a kind of method detecting the relative expression quantity of miRNA in Patients with Osteosarcoma blood plasma or serum, packet are provided Include following steps:
(1) it extracts the total serum IgE of test plasma or serum and reverse transcription is cDNA；
(2) using step (1) obtain cDNA as template, detect respectively wherein the content of the coded sequence of purpose miRNA with The content of the coded sequence of hsa-miR-128a-5p and let-7d；
(3) purpose is calculated as a result, using the hsa-miR-128a-5p and let-7d as reference gene according to step (2) The relative expression quantity of miRNA.
The advantages of serum/plasma miRNA serum s detection internal reference provided by the invention, is:
(1) serum/plasma miRNA serum s is a kind of new biomarkers, is different from traditional biological marker, not only stable, It is minimally invasive, be easy to detect, and it is quantitative accurate, the sensibility and specificity of medical diagnosis on disease will be greatly improved, such microRNA internal reference Successful research facilitate serum/plasma miRNA serum s osteosarcoma disease early diagnosis and in terms of value clinic The comparison of result of study between conversion and different experiments room.
(2) serum/plasma miRNA serum s internal reference detection kit can be used for detecting the internal reference in different experimenter's serum/blood plasma MiRNAs facilitates the baseline expression level for reflecting different subject's internal reference miRNA, causes for controlling qautobiology difference MiRNA expression difference, to highlight really differential expression miRNA relevant to disease.
(3) strict screening and proof scheme are used, the present inventor's initial stage is using genetic chip to osteosarcoma and normal person Serum/plasma miRNA serum s carries out expression analysis, to obtain the serum/plasma miRNA serum s for stablizing expression, and applies qRT-PCR The method of (sonde method and dye method) has carried out single sample verifying；The application of above method and strategy ensure that serum/plasma The accuracy of miRNAs internal reference detection kit.
In short, the combination of hsa-miR-128a-5p or hsa-miR-128a-5p and let-7d is steady in Patients with Osteosarcoma Fixed expression, uses the combination of hsa-miR-128a-5p or hsa-miR-128a-5p and let-7d to may be implemented as reference gene The comparison of result of study between different experiments room promotes the normalized of each experimental result, and then promotes serum/plasma miRNA serum Result of study is converted to clinical success, is provided more accurate diagnosis, parting, Index for diagnosis and individuation for clinician and is controlled The powerful for the treatment of.Using single miRNA or two miRNA combination be used as the reference gene in serum or blood plasma, be suitable for height, The detection platform of small throughput detects larger scale clinical and applies, reduces operation difficulty, simplify operating process.
Specific embodiment
MiRNA expression analysis in 1 osteosarcoma patient's peripheral blood of example
1, sample collection
Osteosarcoma patient's peripheral blood is all from because of osteosarcoma inpatient, 30 primary Patients with Osteosarcoma and 20 health Control.Primary Patients with Osteosarcoma group and healthy control group require at least 12h on an empty stomach, in next morning 7:00~8:00 room temperature Under, 10ml venous blood is extracted in ethylenediamine tetra-acetic acid (EDTA) anticoagulant tube, is extracted peripheral blood mononuclear cells PBMCs, is added 1mlTrizol reagent (Invitrogen company), mixes well, -80 DEG C of preservation samples, to extract for RNA.All blood samples Answered with pathological examination it is true and reliable, research ratify through Ethics Committee, patient's informed consent.
2, method
2.1, the extraction of peripheral blood total serum IgE
The total serum IgE that osteosarcoma patient and Normal human peripheral's haemocyte are extracted by Triazol kit specification, passes through gel Electrophoresis proves the integrality of RNA, with the concentration and purity of nucleic acid-protein instrument measurement RNA.Steps are as follows:
(1) patient's peripheral blood is collected, cell is collected by centrifugation, 1mLTrizol reagent is added in every pipe, mixes, is placed at room temperature for 5min.200 μ L of chloroform is added, with forced oscillation 15sec, is incubated at room temperature 2~3min.
(2) 15min is centrifuged with the speed of 12000 × g at 4 DEG C, mixture be divided into red phenol-chloroform phase (lower phase), The interphase and colourless upper strata aqueous phase of white.Total serum IgE is dispersed in upper strata aqueous phase.
(3) upper strata aqueous phase is transferred in a new Eppendorf pipe, 1mL dehydrated alcohol is added, mixed to precipitate RNA。
(4) it is incubated at room temperature 10min or more, 10min is centrifuged with the speed of 12000 × g at 4 DEG C, it is seen that white RNA is heavy Shallow lake is affixed on test tube bottom wall.
(5) supernatant is abandoned, it is primary with 70% ethanol washing RNA precipitate, 5min is centrifuged with the speed of 7500 × g at 4 DEG C.
(6) supernatant is abandoned, is spontaneously dried in air, is dissolved and is precipitated with 60 μ LDFPC water, -70 DEG C save backup.
(7) total serum IgE integrality is identified: being taken 2 μ LRNA samples at 1.5% agarose gel electrophoresis (60v, 30min), is separated EB is dyed after zone, observation of taking pictures under ultraviolet lamp.
(8) with the concentration and purity of nucleic acid-protein instrument measurement RNA.
2.2 gene chip hybridizations and data analysis
The peripheral blood miRNA for taking 100ng to extract, uses the miRNACompleteLabelingandHyb of agilent company Kit, to specifications step carry out sample label and concentration, using agilent company people miRNA chip of expression spectrum into Row hybridization.After carrying out image scanning using chip scanner, data are imported GeneSpringCX11.0 software and carry out data Standardization and subsequent analysis, the valid data application professional software after chip is normalized analyzes.
The qRT-PCR verifying of miRNA in 2 serum/plasma of embodiment
According to chip hybridization results, the miRNA molecule that selection meets following standard is carried out further using qRT-PCR technology Screening: a) expression in osteosarcoma and control group is at first 80；B) in two groups of stable expression, and without bright between two groups Significant difference is different (p >=0.05).According to the above standard, 6 satisfactory miRNA molecules (including hsa-miR-128a- is selected altogether 5p, let-7d, hsa-miR-100, hsa-miR-210-5p, hsa-miR-222, hsa-miR-425), 6 miRNA molecules Sequence is as shown in table 1.Further, since the reference molecules that U6 is often detected as tissue miRNA, it could in serum to verify it As internal reference, U6 is also used as candidate molecules to be included into screening.By using qRT-PCR technology another above-mentioned 6 miRNA and U6 It is verified in outer one group of subject (including 10 osteosarcoma and 10 controls), has 2 candidate molecules because expression is lower And it is excluded (hsa-miR-210-5p, hsa-mir-222).To remaining 4 molecules (and U6), using Normfinder, GeNorm software carries out expression stability analysis.
Table 1miRNA candidate's internalcontrol sequence
Entire research process implements strict quality control, and each sample continuously detects three times and all detections are all made of blind, Complete in the case where not knowing sample background to avoid bias.Specific step is as follows:
(1) RNA sample is prepared: referring to embodiment 1
(2) sonde method real-time quantitative PCR: ABI kit is used.
A) cDNA is obtained by RNA reverse transcription reaction.The reverse transcription reaction system of sonde method includes 0.10 μ l 100mM DNTPs mixture, 1 μ l reverse transcriptase (50U/ μ L), 2.0 μ l 10X RT Buffers, 0.20 μ l RNA inhibitor and 2.5 (the reverse transcriptase primer mixture of above-mentioned 6 miRNA and U6, every kind of miRNA are added 3/4 μ l and reverse 5 × reverse transcriptase primer of μ l Record primer).The total serum IgE of 9.56 μ l is added.Reaction step is 16 DEG C and is incubated for 30 minutes that 42 DEG C are reacted 30 minutes, and 85 DEG C are incubated for 5 points Clock；
B) q-PCR: being added the dilution of 5 μ l water for cDNA, the cDNA after taking 1 μ l to dilute, be separately added into 0.25 μ l 20 × MicroRNA detection probe (probe of above-mentioned 6 miRNA and U6), 2.5 μ 2 × gene expression of l Master Mix, 1.25 μ l Distilled water, 5 μ l systems carry out q-PCR.Instrument uses 7900 fluorescence quantitative PCR instrument of ABI Prism, the reaction condition of PCR Be: 95 DEG C, 5 minutes progress 1 circulation → 95 DEG C, 15 seconds, 58 DEG C, 1 minute carry out 45 circulations.
(3) dye method real-time quantitative PCR:
A) cDNA is obtained by RNA reverse transcription reaction.The reaction system of the reverse transcription of dye method is slow including 45 × AMV of μ l Fliud flushing, 2 μ l 10mM dNTP mixtures (Takara company), 0.5 μ l RNase inhibitor (Takara company), 2 μ l AMV Mixture (the specificity of above-mentioned 6 miRNA and U6 of (Takara company) and 1.5 μ l miRNA specific reverse primers Reverse primer Mix), every kind of 1.5/4 μ l of addition).Reaction step is 16 DEG C and is incubated for 15 minutes that 42 DEG C are reacted 1 hour, 85 DEG C It is incubated for 5 minutes；
B) q-PCR: pressing 1/5 volume dilution for cDNA, and 0.15 μ l Taq enzyme is added in the cDNA after taking 0.5 μ l to dilute (Takara company), 0.5 20 × EVAGREEN of μ l, 0.1 10 μM of μ l forward primer one kind (are respectively adopted above-mentioned single The corresponding forward primer of miRNA and U6), the general reverse primer (URP:TGGTGTCGTGGAGTCG) of 0.1 10 μM of μ l, 0.6 μ l 25mM MgCl2, 0.8 μ l 2.5mM dNTP mixture (Takara company), 1 μ l 10 × PCR buffer, 6.75 μ lH2O, 10 μ l System carries out q-PCR.Instrument uses 7900 fluorescence quantitative PCR instrument of ABI Prism, and the reaction condition of PCR is: 95 DEG C, 5 Minute carries out 1 circulation → 95 DEG C, 15 seconds, and 58 DEG C, 1 minute carry out 45 circulations.
(4) data process&analysis
To each miRNA molecules of all sample serum, testing result (Ct value) is averaged three times, exclude expression compared with The miRNA molecule of low (Ct value median is greater than 35), analyzes each miRNA using One-wayANOVA method to remaining molecule and exists Whether there is differential expression between different sample groups, (is shown in Table with the stability that Normfinder and geNorm calculate each miRNA 2).As shown in Table 1, according to single miRNA as serum internal reference, hsa-miR-128a-5p is the highest internal reference of stability, and The combinative stability of hsa-miR-128a-5p and let-7d is more preferably.
Therefore, inventors demonstrated that the group that hsa-miR-128a-5p or hsa-miR-128a-5p/let-7d is constituted Conjunction can be expressed steadily in each osteosarcoma patient and normal healthy controls.
The stability analysis of table 2miRNA internal reference
The further verifying of 3 expression stability of embodiment
According to qRT-PCR the selection result, the group of hsa-miR-128a-5p or hsa-miR-128a-5p/let-7d are combined into Candidate reference gene is stablized in expression.Using qRT-PCR method one group of new tested sample (including 10 osteosarcoma and 10 it is right In the same old way originally its stability is verified in), its stable expression in osteosarcoma and normal sample as the result is shown, is had preferable Stability.
The production of 4 serum/plasma miRNA serum internal reference detection kit of embodiment
Kit includes serum/plasma miRNA serum primer (including individual hsa-miR-128a-5p or hsa-miR- The primer of 128a-5p/let-7d combines, common enzyme and/or reagent needed for can also having corresponding PCR reaction, such as: reverse transcription Enzyme, buffer, dNTPs, MgCl2, nuclease water is removed, fluorescent dye or probe, Taq enzyme etc. can be according to the experiments specifically used Method selection, these common enzymes and/or reagent be it is well known to those skilled in the art, in addition it can have standard items and control. The value of this kit is the internal reference of detectable osteosarcoma serum/plasma miRNA serum, the ratio of result of study between different experiments room Compared with foundation is provided, to promote the clinical conversion of correlative study result, therefore this kit is put into and is practiced, can help to instruct to face Bed accurately makes diagnosis and more effective individualized treatment.

Claims (1)

1. a kind of application of combination of miRNA in preparation for the internal reference reagent of osteosarcoma miRNA detection, it is characterised in that: institute It states group and is combined into the combination that hsa-miR-128a-5p and let-7d is constituted, wherein the sequence of hsa-miR-128a-5p are as follows: 5 '- The sequence of CGGGGCCGUAGCACUGUCUGAGA-3 ', let-7d are as follows: 5 '-AGAGGUAGUAGGUUGCAUAGUU-3 '.
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