CN108085386A - The identification of the reference gene of osteosarcoma miRNA detections - Google Patents
The identification of the reference gene of osteosarcoma miRNA detections Download PDFInfo
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Abstract
The invention discloses the internal reference miR-96 gene for stablizing expression in osteosarcoma patient, the combination formed 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 detections, can realize the comparison of result of study between different experiments room, promote the normalized of each experimental result.
Description
Technical field
Patent of the present invention is related to a kind of identification of the internal reference miR-96 gene for osteosarcoma miRNA detections.
Background technology
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 fusiformis 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 attack and transfer of osteosarcoma seriously affect quality of life and the prognosis of patient.Although clinical orthopaedics worker is always
It is directed to the research prevented it, but the overall 5 years survival rates of osteosarcoma still fluctuated in recent years, therefore, searched out bone and flesh
Knurl 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 ripe nucleotide
RNAmolecules, ncRNAs), it is found for the first time when nematode is being studied by Ambros seminars within 1993.So far,
MiRNA exists in all animals and plants, accounts for the 4% of genome.They rise in the generation of cell, differentiation, multiplication and apoptosis
Important role.In addition, based on the basis of target gene function, miRNA has extensive adjustment effect to cancer, they
Tumor suppressor can be used as, can also be used as the tumorigenesis factor.Main mechanism of action includes：The missing in miRNA sites expands
Increase, mutation, epigenetic is horizontal to be changed, and turns unconventionality expression of clever regulatory factor etc..MiRNA is being organized and the expression table in 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 notable.Existing research passes through height at present
Flux is sequenced or biochip technology detects the expression of miRNA to determine with the relevant miRNA of osteosarcoma, including miR-
4714-3p, miR-1299, miR-6779-5p, miR-1292, miR-3688-3p, miR-5010 etc..
There is various factors that can cause sample variation, such as the quality and quantity of RNA in gene expression research, but express
Data can correct this deviation by the correction of reference gene.The selection of miRNA reference genes is extremely important.There is research table
Bright, the selection of reference gene can make a significant impact RT-PCR results, if reference gene selection is incorrect, will make between sample
Gene expression difference is blanked or excessively exaggerates, and causes 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 controls of the mankind (such as nematode) miRNA as miRNA standardized testings, 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 detections
Reference gene, such as 5SrRNA, 18SrRNA and U6 etc., but since these genes are not miRNA, it is impossible to 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 there is no to carry out the optimal reference gene that miRNA in osteosarcoma research is standardized at present
Systematic identification and assessment, therefore, there is an urgent need in the art to develop that microRNA in osteosarcoma research can be used as to standardize
Reference gene, and establish detection miRNA, particularly cycle miRNA effective standard scheme.
The content of the invention
To solve the above problems, the identification of the internal reference miR-96 gene the invention discloses a kind of osteosarcoma miRNA detections.
Specifically, the present invention provides a kind of applications of miRNA preparing the internal reference reagent for osteosarcoma miRNA detections,
It is characterized in that：The miRNA is hsa-miR-128a-5p.
The present invention also provides a kind of combinations of miRNA to prepare for the application of the osteosarcoma miRNA internal reference reagents detected,
It is characterized in that：The combination that the combination is formed for hsa-miR-128a-5p and let-7d.
The wherein sequence of hsa-miR-128a-5p is：The sequence of 5 '-CGGGGCCGUAGCACUGUCUGAGA-3 ', let-7d
It is classified as：5’-AGAGGUAGUAGGUUGCAUAGUU-3’.
The present invention also provides the internal reference reagent detected for osteosarcoma miRNA in the kit for preparing diagnosis osteosarcoma
Using.A kind of kit for diagnosing osteosarcoma is provided, which, which contains, is useful for detection internal reference miRNA hsa-miR-128a-5p
Or the reagent of the combination of hsa-miR-128a-5p and let-7d.
Further, the kit is also included for the required RNA separating liquids of miRNA extractions and qRT-PCR reactions, examination
Agent and enzyme and standard items or/and reference substance.
The present invention also provides a kind of method for detecting the relative expression quantity of miRNA in Patients with Osteosarcoma blood plasma or serum, bags
Include following steps：
(1) it is cDNA to extract the total serum IgE of test plasma or serum and reverse transcription；
(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 for detecting the relative expression quantity of miRNA in Patients with Osteosarcoma blood plasma or serum, bag are provided
Include following steps：
(1) it is cDNA to extract the total serum IgE of test plasma or serum and reverse transcription；
(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 detections 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 stablize,
It is minimally invasive, be easy to detect, it is and quantitative accurate, the sensibility and specificity of medical diagnosis on disease will be greatly improved, such microRNA internal reference
Successful research contribute to serum/plasma miRNA serum s in the early diagnosis of osteosarcoma disease and the clinic of Index for diagnosis etc. value
The comparison of result of study between conversion and different experiments room.
(2) serum/plasma miRNA serum s internal references detection kit can be used for detecting the internal reference in different experimenter's serum/blood plasma
MiRNAs contributes to the baseline expression level for reflecting different subject's internal reference miRNA, for qautobiology difference to be controlled to cause
MiRNA expression differences, with highlight really with the relevant differential expression miRNA of 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 using qRT-PCR
The method of (sonde method and dye method) has carried out single sample verification；The application of above method and strategy ensure that serum/plasma
The accuracy of miRNAs internal reference detection kits.
In short, the combination of hsa-miR-345-5p or hsa-miR-345-5p and hsa-miR-99b is in Patients with Osteosarcoma
Middle stable expression, by the use of the combination of hsa-miR-345-5p or hsa-miR-345-5p and hsa-miR-99b as reference gene
It can realize the comparison of result of study between different experiments room, promote the normalized of each experimental result, and then promote serum/blood
Starch miRNA results of study to clinical success convert, for clinician provide more accurate diagnosis, parting, Index for diagnosis and
The powerful of individualized treatment.Using the combination of single miRNA or two miRNA as the reference gene in serum or blood plasma,
Suitable for the detection platform of high and low flux, detect and apply for larger scale clinical, reduce operation difficulty, simplify operation stream
Journey.
Specific embodiment
MiRNA expression analysis in 1 osteosarcoma patient's peripheral blood of example
1st, 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 requirement empty stomach at least 12h, in m seq 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 in
1mlTrizol reagents (Invitrogen companies), abundant mixing, -80 DEG C of preservation samples extract for RNA.All blood samples
Should be true and reliable with pathological examination, it studies and ratifies through Ethics Committee, patient's informed consent.
2nd, method
2.1st, the extraction of peripheral blood total serum IgE
The total serum IgE of osteosarcoma patient and Normal human peripheral's haemocyte is extracted by Triazol kit specifications, passes through gel
Electrophoresis proves the integrality of RNA, and the concentration and purity of RNA are measured with nucleic acid-protein instrument.Step is as follows：
(1) patient's peripheral blood is collected, cell is collected by centrifugation, often pipe adds in 1mLTrizol reagents, and mixing is placed at room temperature for
5min.200 μ L of chloroform are added in, with forced oscillation 15sec, are 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, adds in 1mL absolute ethyl alcohols, mixing is to precipitate
RNA。
(4) more than 10min is incubated at room temperature, 10min is centrifuged with the speed of 12000 × g at 4 DEG C, it is seen that white RNA sinks
Shallow lake is affixed on test tube bottom wall.
(5) supernatant is abandoned, RNA precipitate is washed once with 70% ethyl alcohol, 5min is centrifuged with the speed of 7500 × g at 4 DEG C.
(6) supernatant is abandoned, is spontaneously dried in air, is precipitated with 60 μ LDFPC water dissolutions, -70 DEG C save backup.
(7) total serum IgE integrality is identified：2 μ LRNA samples is taken to separate at 1.5% agarose gel electrophoresis (60v, 30min)
EB is dyed after zone, observation of taking pictures under ultraviolet lamp.
(8) concentration and purity of RNA is measured with nucleic acid-protein instrument.
2.2 gene chip hybridizations and data analysis
The peripheral blood miRNA that 100ng is taken to extract, uses the miRNAComplete LabelingandHyb of agilent company
Kit, to specifications step carry out sample mark 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 softwares and carry out data
Standardization and subsequent analysis, the valid data application professional software after chip is normalized analyze.
The qRT-PCR verifications 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 technologies
Screening：A) expression in osteosarcoma and control group is at first 80；B) expression is stablized at two groups, and without bright between two groups
Significant difference is different (p >=0.05).According to more than standard, 6 satisfactory miRNA molecules are selected altogether (including hsa-miR-128a-
5p, let-7d, has-miR-100, has-miR-210-5p, has-miR-222, has-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, to verify that it could in serum
As internal reference, U6 also serves as candidate molecules and is included into screening.By using qRT-PCR technologies another to above-mentioned 6 miRNA and U6
Verified there are 2 candidate molecules because expression is relatively low in outer one group of subject (being compareed including 10 osteosarcoma and 10)
And it is excluded (has-miR-210-5p, has-mir-222).To remaining 4 molecules (and U6), using Normfinder,
GeNorm softwares carry out expression stability analysis.
1 miRNA candidate's internalcontrol sequences of table
Entire research process implements strict quality control, and continuously detection is three times and all detections use blind for each sample,
It is completed i.e. in the case where not knowing sample background to avoid bias.It is as follows：
(1) RNA sample is prepared：Referring to embodiment 1
(2) sonde method real-time quantitative PCR：Use ABI kits.
A) cDNA is obtained by RNA reverse transcription reactions.The reverse transcription reaction system of sonde method includes 0.10 μ l 100mM
DNTPs mixtures, 1 μ l reverse transcriptases (50U/ μ L), 2.0 μ l10X RT Buffers, 0.20 μ l RNA inhibitor and 2.5 μ
(the reverse transcriptase primer mixture of above-mentioned 6 miRNA and U6, each miRNA add in 3/4 μ l reverse transcriptions to 5 × reverse transcriptase primers of l
Primer).Add in the total serum IgE of 9.56 μ l.Reaction step is incubated 30 minutes for 16 DEG C, and 42 DEG C are reacted 30 minutes, and 85 DEG C are incubated 5 points
Clock；
b)q-PCR：CDNA is added in into the dilution of 5 μ l water, take 1 μ l dilute after cDNA, be separately added into 0.25 μ l 20 ×
MicroRNA detection probes (probe of above-mentioned 6 miRNA and U6), 2.5 μ 2 × gene expressions of l Master Mix, 1.25 μ l
Distilled water, 5 μ l systems carry out q-PCR.Instrument uses 7900 fluorescence quantitative PCR instruments of ABI Prism, the reaction condition of PCR
It is：It carries out 1 for 95 DEG C, 5 minutes and cycles → 95 DEG C, 15 seconds, 58 DEG C, 1 minute carry out 45 Xun Huans.
(3) dye method real-time quantitative PCR：
A) cDNA is obtained by RNA reverse transcription reactions.The reaction system of the reverse transcription of dye method is delayed including 45 × AMV of μ l
Fliud flushing, 2 μ l 10mM dNTP mixtures (Takara companies), 0.5 μ l RNase inhibitor (Takara companies), 2 μ l AMV
Mixture (the specificity of above-mentioned 6 miRNA and U6 of (Takara companies) and 1.5 μ l miRNA specific reverse primers
Reverse primer Mix), each adds in 1.5/4 μ l).Reaction step is incubated 15 minutes for 16 DEG C, when 42 DEG C of reactions 1 are small, 85 DEG C
It is incubated 5 minutes；
b)q-PCR：By cDNA by 1/5 volume dilution, the cDNA after 0.5 μ l dilutions is taken, adds in 0.15 μ l Taq enzymes
(Takara companies), 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 primers of miRNA and U6), the 0.1 general reverse primer (URP of 10 μM of μ l：TGGTGTCGTGGAGTCG), 0.6 μ l
25mM MgCl2, 0.8 μ l 2.5mM dNTP mixtures (Takara companies), 1 μ l 10 × PCR buffer solutions, 6.75 μ lH2O, 10 μ l
System carries out q-PCR.Instrument uses 7900 fluorescence quantitative PCR instruments of ABI Prism, and the reaction condition of PCR is：95℃、5
Minute carries out 1 and cycles → 95 DEG C, 15 seconds, and 58 DEG C, 1 minute carry out 45 cycles.
(4) data process＆analysis
To each miRNA molecules of all sample serum, testing result (Ct values) is averaged three times, exclude expression compared with
The miRNA molecule of low (Ct values median is more than 35) analyzes each miRNA using One-wayANOVA methods to remaining molecule and exists
Whether there is differential expression between different sample groups, the stability that each miRNA is calculated with Normfinder and geNorm (is shown in Table
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 has-miR-128a-5p or has-miR-128a-5p/let-7d is formed
Conjunction can be expressed steadily in each osteosarcoma patient and normal healthy controls.
The stability analysis of 2 miRNA internal references of table
The further verification of 3 expression stability of embodiment
According to qRT-PCR the selection results, has-miR-128a-5p or has-miR-128a-5p/let-7d's is combined as
Candidate's reference gene is stablized in expression.It is (right including 10 osteosarcoma and 10 in one group of new tested sample using qRT-PCR methods
In the same old way originally its stability is verified in), it stablizes expression to the results show in osteosarcoma and normal sample, has preferable
Stability.
The making of 4 serum/plasma miRNA serum internal reference detection kit of embodiment
Kit includes serum/plasma miRNA serum primer (including individual has-miR-128a-5p or has-miR-
The primer combination of 128a-5p/let-7d can also have common enzyme and/or reagent needed for corresponding PCR reactions, such as：Reverse transcription
Enzyme, buffer solution, 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 are well known to those skilled in the art, it can in addition contain 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, the clinical of correlative study result to be promoted to convert, therefore this kit is put into and is put into practice, can help to instruct to face
Bed accurately makes diagnosis and more effective individualized treatment.
Claims (8)
1. a kind of miRNA is in the application for preparing the internal reference reagent for osteosarcoma patient miRNA detections, it is characterised in that：It is described
MiRNA is hsa-miR-128a-5p.
2. a kind of combination of miRNA is in the application for preparing the internal reference reagent for osteosarcoma miRNA detections, it is characterised in that：Institute
It states and is combined as the combination that hsa-miR-128a-5p and let-7d is formed.
3. application as claimed in claim 1 or 2, the wherein sequence of hsa-miR-128a-5p are：5’-
The sequence of CGGGGCCGUAGCACUGUCUGAGA-3 ', let-7d is：5’-AGAGGUAGUAGGUUGCAUAGUU-3’.
4. the internal reference reagent for osteosarcoma miRNA detections described in claim 1 or 2 is preparing the kit of diagnosis osteosarcoma
In application.
5. a kind of kit for diagnosing osteosarcoma, it is characterised in that：The kit containing be useful for test right requirement 1 or right will
Seek the reagent of the internal reference miRNA described in 2.
6. the kit of diagnosis osteosarcoma according to claim 5, it is characterised in that：The kit is also included and is used for
RNA separating liquids, reagent and the enzyme and standard items or/and reference substance that miRNA is extracted and qRT-PCR reactions are required.
7. a kind of method for detecting the relative expression quantity of miRNA in Patients with Osteosarcoma blood plasma or serum, includes the following steps：
(1) it is cDNA to extract the total serum IgE of test plasma or serum and reverse transcription；
(2) cDNA obtained using step (1) detects the content and hsa- of the wherein coded sequence of purpose miRNA respectively as template
The content of the coded sequence of miR-128a-5p；
(3) the opposite of purpose miRNA is calculated as a result, using the hsa-miR-128a-5p as reference gene according to step (2)
Expression quantity.
8. a kind of method for detecting the relative expression quantity of miRNA in Patients with Osteosarcoma blood plasma or serum, includes the following steps：
(1) it is cDNA to extract the total serum IgE of test plasma or serum and reverse transcription；
(2) cDNA obtained using step (1) detects the content and hsa- of the wherein coded sequence of purpose miRNA respectively as template
The content of the coded sequence of miR-128a-5p, 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.
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