CN106119347A - The primer of colorectal cancer based on serum exosomal microRNAs transfer detection and test kit - Google Patents
The primer of colorectal cancer based on serum exosomal microRNAs transfer detection and test kit Download PDFInfo
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Abstract
The invention discloses a kind of colorectal cancer based on serum exosomal microRNAs transfer detection primer and test kit and, secondary high throughput sequencing technologies is utilized to lymphatic metastasis colorectal cancer patients and not occur lymphatic metastasis serum in patients with colorectal exosomal microRNAs differential expression spectrum to screen, choose up-regulated expression the most obvious miR 483 5p the most respectively and downward is expressed the most obvious miR 451a and carried out large sample checking, find that miR 483 5p, miR 451a has the Differential Diagnosis usefulness of preferable Regional Lymph Nodes of Colorectal Cancer transfer.Meanwhile, present invention uses miR 191 5p, U6 and combine as reference gene, miR 483 5p, miR 451a in specimen is carried out relative quantitative assay, improves data accuracy.The present invention is according to formula E=10(‑1/Slope)1, calculate amplification efficiency (E), according to formula Q=(E+1)‑△CqCalculate miR 483 5p, miR 451a expression relative to miR 191 5p, U6, it is to avoid tradition 2‑△△CqThe restriction that method necessarily requires PCR amplification efficiency to be 100%, makes interpretation of result relatively reliable.
Description
Technical field
The invention belongs to technical field of molecular biological detection, relate to a kind of detection colorectal cancer transfer associated serum
The primer special of exosomal microRNAs, test kit, and detection colorectal cancer serum exosomal microRNAs table
The method of the amount of reaching.
Background technology
Colorectal cancer is the malignant tumor that a kind of M & M is higher, and it is that rectal cancer patient treatment is lost that transfer occurs
Lose and dead main reason.Research display, the colorectal cancer patients exceeding half eventually shifts, and causes postoperative swollen
The transfer and relapse of tumor.From the point of view of clinical therapeutic efficacy, to transfer or the Accurate Prediction of transfer high-risk group, actively take preventative
Remedy measures is reduction colorectal cancer relapse and metastasis, improve the important step of prognosis.It is used for clinically examining colorectal cancer at present
The method of transfer is more, though respectively having advantage, but clinical practice is all subject to certain restrictions.Intestinal mirror can determine that colorectal cancer patients art
Rear tumor is with or without recurrence and the infiltration degree of recurrence stove, but operates for invasive, and the toleration of patient is poor.Imaging examination side
Method, such as B ultrasonic, CT, MRI and PET/CT etc., although technology is evolving, and sensitivity is the most relatively low, and time to be found, tumor is many has
Standby certain volume.At present, Chinese scholars is tended to use the method for detection tumor markers to shift for colorectal cancer more
Prediction.Therefore, find sensitivity height, the biomarker of high specificity effectively predicts that colorectal cancer transfer is also always clinic and grinds
The focus studied carefully.
MicroRNAs is the endogenous non-coding microRNA that a class has adjusting function, controls the mankind of about 1/3
The expression of gene, it can play the function being similar to oncogene or antioncogene, tumor generation, develop and shift in send out
Wave important function.Nicoloso etc. are at " Nature Reviews Cancer " (Nicoloso, MS.et al (2009) Nat
Rev Cancer 9:293-302) in point out, microRNAs is a kind of novel tumor transfer modulator, as judging that tumor turns
The molecular marker moved is the most potential.MicroRNAs can be by promoting the infiltration of tumor cell, angiogenesis, EMT
With escape apoptosis, participate in the transfer of colorectal cancer.
The U.S. " Popular Science " magazine in 2014 proposes, " with the Exosomes (secreting outward body) the diagnosis work as carrier
Tool, is expected to make the cancer diagnosis simpler easy ".Exosomes refers to be divided by cell secret be discharged into extracellular a kind of film capsule
Bubble, plays key player in the invasion and attack, transfer of tumor.(Huang, X.et al (2013) the BMC genomics such as Huang
14:319) utilize degree of depth sequencing analysis to find in blood plasma exosomes and there is abundant RNAs, and the film capsule of exosomes
Bubble structure can produce protective effect, therefore compared with serum/plasma general RNA, serum/plasma exosomal to these RNAs
RNA is more stable, and more can represent the change of the biological function of malignant tumor.In recent years, relevant exosomal RNA
Research for tumor disease diagnostic effect is increasing, for the detection analysis of tumor provide a visual windows (Taylor,
DD.et al (2013) Frontiers in genetics 4:142), but up to the present, relevant to colorectal cancer transfer
Exosomal microRNAs has no report.In addition the heterogeneity between different tumors is very big, relevant to other neoplasm metastasis
Exosomal microRNAs is little to the referential of colorectal cancer transfer detection.Therefore, it is necessary to colorectal cancer is shifted phase
The exosomal microRNAs closed carries out screening verification, finds the exosomal that can be used for colorectal cancer transfer detection
microRNAs。
Summary of the invention
For above-mentioned prior art, the invention provides a kind of detection colorectal cancer transfer associated serum accurate, reliable
The reverse transcriptase primer of exosomal microRNAs and detection primer, detection kit, and detection colorectal cancer serum
The method of exosomal microRNAs expression.
The present invention is achieved by the following technical solutions:
The reagent of detection miR-483-5p Yu miR-451a combination is examined based on serum exosomal microRNAs in preparation
Survey the application in colorectal cancer transfering reagent, wherein the base sequence of miR-483-5p, miR-451a be respectively 5 '-
AAGACGGGAGGAAAGAAGGGAG-3’(SEQ ID:1)、5’-AAACCGUUACCAUUACUGAGUU-3’(SEQ ID:2)。
A kind of primer based on serum exosomal microRNAs detection colorectal cancer transfer, including miR-483-5p's
Reverse transcriptase primer and detection primer, the reverse transcriptase primer of miR-451a and detection primer,
Reverse transcriptase primer and the detection primer of miR-483-5p are as follows:
MiR-483-5p-RT:
5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTCCCTTC-3’(SEQ ID:3)；
MiR-483-5p-F:5 '-ACACTCCAGCTGGGAAGACGGGAGGAAAGA-3 ' (SEQ ID:4)；
MiR-483-5p-R:5 '-TGGTGTCGTGGAGTCG-3 ' (SEQ ID:5)；
Reverse transcriptase primer and the detection primer of miR-451a are as follows:
MiR-451a-RT:
5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAACTCAGT-3’(SEQ ID:6)；
MiR-451a-F:5 '-ACACTCCAGCTGGGAAACCGTTACCATTAC-3 ' (SEQ ID:7)；
MiR-451a-R:5 '-TGGTGTCGTGGAGTCG-3 ' (SEQ ID:8).
A kind of test kit based on serum exosomal microRNAs detection colorectal cancer transfer, including above-mentioned miR-
The reverse transcriptase primer of 483-5p and detection primer, the reverse transcriptase primer of miR-451a and detection primer, also include reference gene
MiR-191-5p reverse transcriptase primer, reference gene miR-191-5p forward primer, reference gene miR-191-5p downstream primer, interior
Ginseng gene U6 reverse transcriptase primer, reference gene U6 forward primer, reference gene U6 downstream primer and PCR reactant liquor.
Reverse transcriptase primer and the detection primer of miR-191-5p are as follows:
MiR-191-5p-RT:
5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCAGCTGCT-3’(SEQ ID:9)；
MiR-191-5p-F:5 '-ACACTCCAGCTGGG CAACGGAATCCCAAAAG-3 ' (SEQ ID:10)；
MiR-191-5p-R:5 '-TGGTGTCGTGGAGTCG-3 ' (SEQ ID:11)；
Reverse transcriptase primer and the detection primer of U6 are as follows:
U6-RT:5 '-AACGCTTCACGAATTTGCGT-3 ' (SEQ ID:12)；
U6-F:5 '-CTCGCTTCGGCAGCACA-3 ' (SEQ ID:13)；
U6-R:5 '-AACGCTTCACGAATTTGCGT-3 ' (SEQ ID:14).
Preferably: PCR reactant liquor is by Syber Green I fluorescent dye, archaeal dna polymerase, dNTPs, trishydroxymethylaminomethane
Hydrochlorate, potassium chloride, magnesium chloride and water composition.
Preferably: in described test kit, the concentration of each material is as follows: the reverse transcriptase primer of miR-483-5p: 10nM；miR-
The forward primer of 483-5p: 10nM；The downstream primer of miR-483-5p: 10nM；The reverse transcriptase primer of miR-451a: 10nM；
The forward primer of miR-451a: 10nM；The downstream primer of miR-451a: 10nM；Syber Green I fluorescent dye: 1 × Syber
Green I fluorescent dye；Reference gene miR-191-5p reverse transcriptase primer: 10nM；Reference gene miR-191-5p forward primer:
10nM；Reference gene miR-191-5p downstream primer: 10nM；Reference gene U6 reverse transcriptase primer: 10nM；Reference gene U6 upstream
Primer: 10nM；Reference gene U6 downstream primer: 10nM；Archaeal dna polymerase: 100U/mL；DNTPs:0.2mM；Magnesium chloride: 6mM；Three
Hydroxymethyl aminomethane hydrochlorate: 16.5mM；Potassium chloride: 89.3mM.
Above-mentioned primer based on serum exosomal microRNAs detection colorectal cancer transfer, based on serum exosomal
The test kit of microRNAs detection colorectal cancer transfer is at qualitative or quantitative detection serum exosomal microRNAs expression
In application.
A kind of method detecting the relevant exosomal microRNAs expression of colorectal cancer transfer, step is as follows:
(1) exosomes in serum sample is extracted: take 250 μ l serum and add 63 μ l ExoQuickTM
ExosomePrecipitation Solution reagent, concussion mixing, hatch 30min at room temperature.4 DEG C, 1500g, centrifugal
30min, pale yellow precipitate seen from the bottom of pipe, abandon supernatant, by the 25 μ resuspended precipitations of l PBS, if precipitation is insoluble, can suitably continue
Add PBS until being completely dissolved.
(2) total serum IgE in exosomes is separated:
(3) RT-qPCR amplification: the RNA of above-mentioned separation being carried out reverse transcription and becomes cDNA, taking cDNA is template, adds primer
With PCR reactant liquor, carry out PCR reaction, detect sample threshold Cq；Extract Human colorectal cancer cells strain SW480 (purchased from China simultaneously
Academy of Medical Sciences Institute of Basic Medical Sciences preclinical medicine cell centre) RNAs, reverse transcription become cDNA as check and correction sample, each
PCR Sptting plate detects, records Cq value；The RT-qPCR amplification of reference gene miR-191-5p, U6, except primer uses phase
Outside corresponding reverse transcriptase primer and detection primer, method is identical；
(4) standard curve is made: after above-mentioned detection completes, copy number is taken the logarithm using 10 the end of for as abscissa, and Cq value is
Vertical coordinate is mapped, and draws standard curve, and slope calculations Slope, then according to formula E=10(-1/Slope)-1, calculate amplification efficiency
E；
(5) calculating: choose sample and check and correction pattern detection hole, the accompanying software of application real-time fluorescence quantitative PCR instrument draws
Sample threshold Cq (T) and check and correction sample threshold Cq (C), according to formula Q=(E+1)-△Cq, △ Cq=[Cq (T)-Cq (C)], draw
The correction starting copy number Q of gene；Q by the Q-value of miR-483-5p, miR-451a Yu reference gene (miR-191-5p, U6)
The geometric mean of value is compared, and obtains the relative expression quantity of miR-483-5p, miR-451a.
The purpose of said method is the application in the change of research malignant tumor biological function, is not examining with disease
Break and for the purpose for the treatment of.
Beneficial effect:
The exosomal microRNAs detection method that the present invention sets up, effectively judging and controlling for colorectal cancer transfer
The selection for the treatment of scheme provides important reference frame.
Present invention uses miR-191-5p, U6 combination as internal reference to miR-483-5p, the miR-451a in specimen
Carrying out relative quantitative assay, through NormFinder, geNorm software evaluation, stability is better than single reference gene miR-191-5p
Or the use of U6, improve the accuracy of result, be shown in Table 1.
Table 1NormFinder and the evaluation to reference gene of the geNorm software
Note: marking the lowest, stability is the best.
The present invention is according to formula E=10(-1/Slope)-1, calculate amplification efficiency (E), according to formula Q=(E+1)-△CqCalculate
MiR-483-5p, miR-451a gene expression relative to miR-191-5p, U6, it is to avoid tradition 2-△△CqMethod necessarily requires PCR
Amplification efficiency is the restriction of 100%, makes interpretation of result relatively reliable.
Accompanying drawing explanation
Fig. 1 is lymphatic metastasis colorectal cancer patients and lymphatic metastasis serum in patients with colorectal exosomal does not occurs
MicroRNAs differential expression is composed；
Fig. 2 is the standard curve of miR-191-5p, U6, miR-483-5p and miR-451a；
Fig. 3 is the Evaluation on specificity of test kit detection miR-483-5p, miR-451a；
Fig. 4 is that exosomal miR-483-5p, miR-451a do not occur lymphatic metastasis colorectal cancer (NLNM) in 47 examples
With the relative expression levels in 69 examples lymphatic metastasis colorectal cancer (LNM)；
Fig. 5 is that exosomal miR-483-5p, miR-451a detect the ROC curve judging that colorectal cancer shifts.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further illustrated.
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Embodiment 1
(1) screening of the relevant exosomal microRNAs of colorectal cancer transfer:
Use " microRNAs cDNA library+library expands+secondary order-checking in advance " research strategy, initially set up based on
The Technology Ways of differential expression microRNAs in serum exosomes, to 40 example lymphatic metastasis colorectal cancer patients (every 20 examples
Divide 1 group into) and 40 examples there is not lymphatic metastasis colorectal cancer patients (every 20 examples divide 1 group into) serum exosomal
MicroRNAs express spectra has carried out analysis and comparison, finds that 13 are raised exosomal microRNAs, wherein miR-483-5p
Up-regulated expression is the most obvious, and 106 are lowered exosomal microRNAs, and wherein miR-451a lowers and expresses the most substantially, sees
Fig. 1.
(2) composition of test kit and preparation:
The sequence of described primer is the nucleoside reported according to microRNA data base (http://www.mirbase.org/)
Acid sequence miR-483-5p (MIMAT0004761), miR-451a (MIMAT0001631) are template autonomous Design, its primer
Sequence, Tm value are shown in Table 1.
Table 2 primer sequence, Tm value
Described dedicated kit also includes on reference gene miR-191-5p reverse transcriptase primer, reference gene miR-191-5p
Trip primer, reference gene miR-191-5p downstream primer, reference gene U6 reverse transcriptase primer, reference gene U6 forward primer, interior
Ginseng gene U6 downstream primer and PCR reactant liquor, PCR reactant liquor by Syber Green I fluorescent dye, archaeal dna polymerase, dNTPs,
Tri(Hydroxymethyl) Amino Methane Hydrochloride, potassium chloride, magnesium chloride and water composition.
Described miR-483-5p reverse transcriptase primer: 10nM.
Described miR-483-5p forward primer: 10nM.
Described miR-483-5p downstream primer: 10nM.
Described miR-451a reverse transcriptase primer: 10nM.
Described miR-451a forward primer: 10nM.
Described miR-451a downstream primer: 10nM.
Described reference gene miR-191-5p reverse transcriptase primer: 10nM.
Described reference gene miR-191-5p forward primer: 10nM.
Described reference gene miR-191-5p downstream primer: 10nM.
Described reference gene U6 reverse transcriptase primer: 10nM.
Described reference gene U6 forward primer: 10nM.
Described reference gene U6 downstream primer: 10nM.
Described Syber Green I fluorescent dye: 1 × Syber Green I fluorescent dye.
The described archaeal dna polymerase final concentration of 100U/ml in qPCR reactant liquor.
The described dNTPs final concentration of 0.2mM in qPCR reactant liquor.
The described magnesium chloride final concentration of 6mM in PCR reactant liquor.
The described Tri(Hydroxymethyl) Amino Methane Hydrochloride final concentration of 16.5mM in PCR reactant liquor.
The described potassium chloride final concentration of 89.3mM in PCR reactant liquor.
(3) case-data:
The colorectal cancer that in January, 2009 in January, 2010 carries out radical resection for colorectal cancer in Shandong Qilu Hospital is suffered from
Person 116 example, wherein lymphatic metastasis group patient 69 example, male 39 examples, female 30 example, age 23～75 years old, the median age 52 years old；Do not send out
Raw lymphatic metastasis group patient 47 example, male 25 examples, female 22 example, age 25～76 years old, the median age 54 years old, all patients are all through group
Knit pathology to make a definite diagnosis, and be ID, do not accept any treatment before.Between two groups, sex, age comparing difference are without statistics
Meaning (P > 0.05), has comparability.
(4) serum specimen collection: serum collection is in coagulant pipe, and 4 DEG C of 1 600 × g are centrifuged 10min, further 4 DEG C
16000 × g is centrifuged 10min, collects serum, put-80 DEG C frozen to be measured.
(5) separation of exosomes in serum: take 250 μ l serum and add 63 μ lExoQuickTM
ExosomePrecipitation Solution reagent, concussion mixing, hatch 30min at room temperature.4 DEG C, 1500g, centrifugal
30min, pale yellow precipitate seen from the bottom of pipe, abandon supernatant, by the 25 μ resuspended precipitations of l PBS, if precipitation is insoluble, can suitably continue
Add PBS until being completely dissolved.
(6) in exosomes, RNA extracts:
1) in the exosome sample extracted, add 700 μ l QIAzolLysis Reagent, concussion mixing
1min, room temperature is static hatches 5min.
2) add 140 μ l chloroform, build lid, acutely shake mixing.
3), after the static 2-3min of room temperature, centrifuge tube is placed in refrigerated centrifuge, 4 DEG C, 12000g, centrifugal 15min.
4) it is slowly withdrawn centrifuge tube after centrifugal end, upper strata aqueous phase (about 350 μ l) is transferred in new centrifuge tube, notes not
Mesophase to be drawn onto, adds 525 μ l dehydrated alcohol, fully mixes with sample loading gun.
5) draw 700 μ l samples to join in Rnease Mini column and build lid, pillar is put into 2ml centrifuge tube
In, 4 DEG C, 10000g, centrifugal 15s, abandon the liquid being centrifuged out.
6) in pillar, add 700 μ l Buffer RWT, build lid, 4 DEG C, 10000g, centrifugal 15s, abandon and be centrifuged out
Liquid.
7) in pillar, add 500 μ l Buffer RPE build lid, 4 DEG C, 10000g, centrifugal 15s, abandon and be centrifuged out
Liquid.
8) in pillar, add 500 μ l Buffer RPE build lid, 4 DEG C, 10000g, centrifugal 15s, abandon and be centrifuged out
Liquid.
9) pillar taking-up is placed in new centrifuge tube, 17000g, centrifugal 1min, removes the liquid in pillar as far as possible.
10) pillar is placed in new centrifuge tube, thinks pillar adds 30-50 μ l DEPC water (being directly added drop-wise on film),
10000g, centrifugal 1min, elute RNA.RNA is present in liquid phase at the bottom of pipe.
(7) RT-qPCR: use the One Step of Takara companymiRNA cDNA Synthesis
Kit Reverse Transcriptase kit carries out reverse transcription to above-mentioned total serum IgE and becomes cDNA, and taking 5 μ l cDNA is template, carries out qPCR reaction.
QPCR reaction system:
Template DNA: 5ul
PCR reactant liquor: 12.5 μ l
Forward primer (10 μMs): 1 μ l
Downstream primer (10 μMs): 1 μ l
Sterilized water: 5.5 μ l
Reaction condition is: 37 DEG C → 20 minutes, 95 DEG C → 10 minutes；(95 DEG C 15 seconds, 60 DEG C 1 minute) → 40 circulations.
The RT-qPCR amplification of reference gene miR-191-5p, U6, except primer uses corresponding reverse transcriptase primer and detection
Outside primer, method is ibid.
(8) standard curve making: the RNA that will extract from Human colorectal carcinoma SW480 cell, reverse transcription becomes cDNA, then
10 times of gradient dilutions become 5 concentration, as standard substance, together carry out RT-qPCR amplification with sample to be tested, make standard curve.
(9) result judges:
1) copy number is taken the logarithm as abscissa using 10 the end of for, and Cq value is mapped for vertical coordinate, draws standard curve, calculates tiltedly
Rate Slope, then according to formula E=10(-1/Slope)-1, calculate amplification efficiency E；
2) choosing sample and check and correction pattern detection hole, the accompanying software of application real-time fluorescence quantitative PCR instrument draws sample threshold
Value Cq (T) and check and correction sample threshold Cq (C), according to formula Q=(E+1)-△Cq, △ Cq=[Cq (T)-Cq (C)], draw gene
Correction starting copy number Q；Geometry by the Q-value of miR-483-5p, miR-451a Yu the Q-value of reference gene miR-191-5p, U6
Average is compared, and obtains the relative expression quantity of miR-483-5p, miR-451a.
(10) testing result:
1) test kit sensitivity assessment
The RNA concentration extracted from Human colorectal carcinoma SW480 cell is adjusted to 1000ng/ μ l, spends RNase sterilized water
Carry out 10 times of gradient dilutions, obtain 100ng/ μ l (101Times dilution), 10ng/ μ l (102Times dilution), 1ng/ μ l (103Dilution again),
0.1ng/μl(104Times dilution), 0.01ng/ μ l (105Dilution again) variable concentrations sample, respectively detection reference gene miR-191-
5p, U6 and target gene miR-483-5p, miR-451a.Result from Figure 2 it can be seen that from 1000ng/ μ l (0 times of dilution) sample to
0.1ng/μl(104Dilution again) sample, the Cq value that miR-191-5p, U6, miR-483-5p, miR-451a detect presents well
Linear relationship, coefficient R2It is all higher than 0.99；And 0.01ng/ μ l (105Times dilution) sample is not detected by corresponding Cq value,
Show the minimum RNA sample that can detect that concentration is 0.1ng/ μ l of this test kit sensitivity.
2) test kit Evaluation on specificity
Select Human colorectal carcinoma SW480 cell, (miR-483 lowers thin the SW480 cell of downward expression miR-483-5p
Born of the same parents) and lower the SW480 cell (miR-451a lowers cell) expressing miR-451a, the RNA of extraction, simultaneously with water as template
Substitute RNA sample as blank.Use this test kit to above-mentioned four kinds of sample detection, express miR-found that lower
MiR-483-5p, miR-451a water that the SW480 cell detection of miR-451a arrives is expressed in SW480 cell and the downward of 483-5p
Flat significantly lower than SW480 cell, blank is not detected by the expression of miR-483-5p, miR-451a simultaneously, sees that Fig. 3 shows this
Test kit has good specificity.
3) test kit amplification efficiency
Copy number is taken the logarithm using 10 the end of for as abscissa, the Cq of miR-191-5p, U6, miR-483-5p, miR-451a
It is worth as vertical coordinate, makes standard curve and see Fig. 2, calculate miR-191-5p, U6, miR-483-5p, miR-451a and expanded
Efficiency (E) is respectively 0.937,0.952,0.949,0.925, is all higher than 0.9, shows that this test kit amplification efficiency is preferable.
4) there is not lymphatic metastasis serum in patients with colorectal in 69 example lymphatic metastasis colorectal cancer patients and 47 examples
Exosomal miR-483-5p, miR-451a testing result is shown in Fig. 4.Lymphatic metastasis group serum exosomal miR-483-5p
Median levels is respectively 0.851 (0.551～1.120), hence it is evident that higher than do not occur lymphatic metastasis group 0.339 (0.134～
0.521), difference has statistical significance (P < 0.001).Lymphatic metastasis group serum exosomal miR-451a Median levels
It is respectively 0.201 (0.127～0.284), hence it is evident that be less than 0.408 (0.309～0.609) that lymphatic metastasis group does not occurs, poor
Different have statistical significance (P < 0.001).
5), the differential diagnosis value that Regional Lymph Nodes of Colorectal Cancer is shifted by serum exosomal miR-483-5p, miR-451a
Analyze
Using 69 example lymphatic metastasis colorectal cancer patients as positive group, 47 examples do not occur lymphatic metastasis colorectal cancer to suffer from
Person as negative group, Receiver operating curve (the receiver operating of MedCalc 9.3.9.0 statistical software
Characteristic curve, ROC) analyze, the mirror that Regional Lymph Nodes of Colorectal Cancer is shifted by serum exosomal miR-483-5p
Other diagnostic is 0.821 (95%CI 0.739～0.886, Z=8.414, P < 0.001)；Serum exosomal miR-451a
To the Differential Diagnosis usefulness of Regional Lymph Nodes of Colorectal Cancer transfer be 0.803 (95%CI 0.719～0.871, Z=6.953, P <
0.001), Fig. 5 is seen.
Although the detailed description of the invention of the present invention is described by the above-mentioned accompanying drawing that combines, but not the present invention is protected model
The restriction enclosed, one of ordinary skill in the art should be understood that on the basis of technical scheme, and those skilled in the art are not
Need to pay various amendments or deformation that creative work can make still within protection scope of the present invention.
Claims (9)
1. the reagent of detection miR-483-5p Yu miR-451a combination detects based on serum exosomal microRNAs in preparation
Application in colorectal cancer transfering reagent, wherein the base sequence of miR-483-5p, miR-451a is respectively such as SEQ ID:1, SEQ
ID:2。
2. a primer based on serum exosomal microRNAs detection colorectal cancer transfer, inverse including miR-483-5p
The downstream of forward primer sequence such as the SEQ ID:4, miR-483-5p of the sequence of transcription primers such as SEQ ID:3, miR-483-5p
The forward primer sequence of sequence such as the SEQ ID:6, miR-451a of the reverse transcriptase primer of primer sequence such as SEQ ID:5, miR-451a
Row are such as the downstream primer sequence such as SEQ ID:8 of SEQ ID:7, miR-451a.
3. a test kit based on serum exosomal microRNAs detection colorectal cancer transfer, is characterized in that: include
The forward primer sequence such as SEQ ID:4 of the sequence of the reverse transcriptase primer of miR-483-5p such as SEQ ID:3, miR-483-5p,
Sequence such as the SEQ ID:6, miR-of the reverse transcriptase primer of downstream primer sequence such as the SEQ ID:5, miR-451a of miR-483-5p
The downstream primer sequence such as SEQ ID:8, reference gene miR-of forward primer sequence such as the SEQ ID:7, miR-451a of 451a
191-5p reverse transcriptase primer sequence such as SEQ ID:9, reference gene miR-191-5p forward primer sequence such as SEQ ID:10, internal reference
Gene miR-191-5p downstream primer sequence such as SEQ ID:11, reference gene U6 reverse transcriptase primer sequence such as SEQ ID:12, interior
Ginseng gene U6 forward primer sequence such as SEQ ID:13, reference gene U6 downstream primer sequence such as SEQ ID:14 and PCR reactant liquor.
4. test kit as claimed in claim 3, is characterized in that: described PCR reactant liquor by Syber Green I fluorescent dye,
Archaeal dna polymerase, dNTPs, Tri(Hydroxymethyl) Amino Methane Hydrochloride, potassium chloride, magnesium chloride and water composition.
5. test kit as claimed in claim 3, is characterized in that: the concentration of each material is as follows: the reverse transcription of miR-483-5p draws
Thing: the forward primer of 10nM, miR-483-5p: the downstream primer of 10nM, miR-483-5p: 10nM；The reverse transcription of miR-451a
Primer: the forward primer of 10nM, miR-451a: the downstream primer of 10nM, miR-451a: 10nM；Reference gene miR-191-5p
Reverse transcriptase primer: 10nM, reference gene miR-191-5p forward primer: 10nM, reference gene miR-191-5p downstream primer:
10nM；Reference gene U6 reverse transcriptase primer: 10nM, reference gene U6 forward primer: 10nM, reference gene U6 downstream primer:
10nM。
6. test kit as claimed in claim 4, is characterized in that: the concentration of each material is as follows: 1 × Syber Green I fluorescence
Dyestuff, archaeal dna polymerase: 100U/mL；DNTPs:0.2mM；Magnesium chloride: 6mM；Tri(Hydroxymethyl) Amino Methane Hydrochloride: 16.5mM；
Potassium chloride: 89.3mM.
7. primer based on serum exosomal microRNAs detection colorectal cancer transfer or right described in claim 2 are wanted
Ask the arbitrary described test kit based on serum exosomal microRNAs detection colorectal cancer transfer of 3-6 qualitatively or quantitatively
Application in detection serum exosomal microRNAs expression.
8. the method detecting miR-483-5p, miR-451a expression, is characterized in that: step is as follows:
(1) exosomes in serum sample is extracted；
(2) total serum IgE in exosomes is separated:
(3) RT-qPCR amplification: the RNA that step (2) separates being carried out reverse transcription and becomes cDNA, taking cDNA is template, adds right and wants
Ask the primer described in 2 and the PCR reactant liquor described in claim 4 or 6, carry out PCR reaction, detect sample threshold Cq；Carry simultaneously
Taking Human colorectal cancer cells strain SW480RNAs, reverse transcription becomes cDNA as check and correction sample, examines in each PCR Sptting plate
Survey, record Cq value；The RT-qPCR amplification of reference gene miR-191-5p, U6；
(4) standard curve is made: after step (3) has detected, copy number is taken the logarithm using 10 the end of for as abscissa, and Cq value is vertical
Coordinate is mapped, and draws standard curve, and slope calculations Slope, then according to formula E=10(-1/Slope)-1, calculate amplification efficiency E；
(5) calculating: choose sample and check and correction pattern detection hole, the accompanying software of application real-time fluorescence quantitative PCR instrument draws sample
Threshold value Cq (T) and check and correction sample threshold Cq (C), according to formula Q=(E+1)-△Cq, △ Cq=[Cq (T)-Cq (C)], draw gene
Correction starting copy number Q；By the Q-value of miR-483-5p, miR-451a compared with the geometric mean of the Q-value of reference gene,
Obtain the relative expression quantity of miR-483-5p, miR-451a.
9. method as claimed in claim 8, is characterized in that: concretely comprising the following steps of described step (1): takes 250 μ l serum and adds
63μl ExoQuickTMExosomePrecipitation Solution reagent, concussion mixing, hatch 30min at room temperature, 4
DEG C, 1500g, centrifugal 30min, abandon supernatant, resuspended until precipitating with 25 μ l PBS.
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