CN110229907A - Diagnosing gastric cancer and prognosis evaluation blood circulation miRNA biomarker detection kit - Google Patents
Diagnosing gastric cancer and prognosis evaluation blood circulation miRNA biomarker detection kit Download PDFInfo
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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Abstract
The invention belongs to kit technical fields, disclose a kind of diagnosing gastric cancer and prognosis evaluation blood circulation miRNA biomarker detection reagent, the diagnosing gastric cancer and prognosis evaluation blood circulation miRNA biomarker detection reagent detection method include: the processing of serum sample, take a blood sample, are centrifuged, save backup；The extraction of total serum IgE, the total serum IgE extracted are put in -80 DEG C of refrigerators and save backup；Absorbance detection is carried out, the quality of RNA is judged using RNA denaturing formaldehyde gel electrophoresis；It carries out reverse transcription and obtains cDNA, obtained cDNA is subjected to fluorogenic quantitative detection；Testing result is analyzed using relative quantification method.The present invention and traditional tumour marker CEA, CA199, efficiency of the CA724 in diagnosis is assessed, better than traditional tumour marker, the detection of miR-30c is suitable as the diagnosis marker for gastric cancer, and be diagnosing gastric cancer exploitation be new lesion detection marker diagnostic kit, be used for therapeutic evaluation and Index for diagnosis, have a good application prospect.
Description
Technical field
The invention belongs to kit technical field more particularly to a kind of diagnosing gastric cancer and prognosis evaluation blood circulation miRNA
Biomarker detection kit.
Background technique
Currently, the prior art commonly used in the trade is such that
Gastric cancer is digestive system common cancer, and in China, incidence gastric cancer rate and the death rate are higher.It analyzes according to statistics,
China's incidence gastric cancer rate in 2012 is 31.28/10 ten thousand, and the death rate is 22.04/10 ten thousand.Incidence gastric cancer is mostly without obvious specificity
Clinical manifestation and prognosis mala, five year survival rate only have 20-30%, therefore, find gastric cancer serum Specific marker and are used for stomach
The early diagnosis and treatment of cancer have great importance.The diagnosis of traditional tumour marker CEA, CA199, CA724 in gastric cancer
Specificity is not high with sensitivity.For example CEA synthesising part is in the stomach and intestine and blood of fetus, the main table in colorectal cancer patients
It reaches, other tumours include gastric cancer, lung cancer, lung cancer, liver cancer, breast cancer, can also be expressed in the tumours such as cancer of pancreas, non-malignant tumors disease
Become such as renal failure, pulmonary emphysema, rectal polyp, CEA expression can increase when colitis, also suffer from other factors influence, such as smoke
With saliva contamination.MicroRNA is the endogenous non-coding microRNA that a kind of length is about 22 nucleotide, can by with target
Mark mRNA 3 ' holds noncoding region, and complementary combine causes target mrna degradation or inhibits its translation completely or partially, thus to target gene
Carry out post-transcriptional control.MicroRNA plays important function in tumour is formed as oncogene or tumor suppressor gene,
MicroRNA is related to a series of increment that knubble biological processes include tumour, invades, transfer.
And small nucleic acid is a kind of endogenic non-coding RNA found in eucaryote, it is highly conserved in evolution,
Stable and fine adjusting is carried out to the expression of gene in post-transcriptional level and with regulation target-complementary pairing.Due to
It is expressed in miRNA blood, due to the shorter only 19-22 nucleotide of its segment, and the protection by RISC complex, it is not degradable
Stability with height keeps stable after tested in the environment of the acute variations such as multigelation and pH value and is able to maintain surely
It is fixed, therefore be a kind of ideal tumor markers.
MiRNA variation and the substantial connection of tumour have obtained the common recognition of scientist, form the hot spot of a research.Stomach
Cancer is high-incidence one of the malignant tumour in the whole world, and wherein the gastric cancer of one third has belonged to advanced stage in discovery, loses surgical radical treatment
Chance, even the gastric cancer of surgical radical treatment, local recurrence or DISTANT METASTASES IN occur for 60%-70%.The clinical every increasing of early diagnostic rate
Add 1%, the whole world just there are about 760,000 people from death, and there are about 160,000 people can get new life for China, can retrieve number with 1,000,000,000 loss.
Therefore, the development of early diagnosis technology is promoted, there is very urgent economy and society meaning.The express spectra of miRNA and heredity
Expression pattern may will develop into new Biomarkers, help to realize early diagnosis, source parting and the prognosis of tumour
Judgement.MiRNA is mainly the expression that gene is adjusted in the level of mRNA, and adjustment mechanism is ingenious, therefore miRNA is used for gene function
It can study, drug target verifying, gene silencing, purposive gene expression regulation are even used for gene therapy, all have wide
Prospect.
In conclusion problem of the existing technology is: traditional tumour marker CEA, CA199, CA724 is in gastric cancer
Specificity is not high with sensitivity.
Solve the difficulty of above-mentioned technical problem:
Due to tumor markers CEA, the non-specificity of CA199, CA724 synthesising part in vivo causes it a variety of swollen
It is expressed in tumor tissue.For example, CEA is in other non-malignant tumors lesion such as renal failures, pulmonary emphysema, rectal polyp, CEA table when colitis
Up to that can increase, also suffering from smoking and saliva contamination factor influences and expresses and increase.CEA expression is protein, due to it
The reason of own biological characteristic, protein is not sufficiently stable, and preservation is improper degradable, and leads to this kind of tumor markers specificity
The not high reason with sensitivity can not overcome its drawback in clinical detection.Therefore continual exploitation more preferably tumor-marker is needed
Object.
Solve the meaning of above-mentioned technical problem:
Sensitivity and the specificity of detection will be improved as the molecular marked compound of tumor markers by finding better matters,
The foundation effectively treated is provided to clinician.Ideal tumor markers one have a characteristic that
1. it must be generated by malignant cell, and can be surveyed in blood, tissue fluid, juice or tumor tissues
Out；
2. it should not be present in normal tissue or measure in benign disease；
Come 3. a certain tumor marker should be changed into the most patient of the tumour to measure；
4. preferably clinically it can still detect before no evidence of tumor, i.e., it is sensitiveer than existing iconography means；
5. the amount of tumor marker is preferably positively correlated with the size of tumour；
6. can help to assessment curative effect to a certain extent, the recurrence and transfer of tumour are predicted.
Summary of the invention
In view of the problems of the existing technology, the present invention provides diagnosing gastric cancer and prognosis evaluation blood circulation miRNA are raw
Object marker detection kit.
The invention is realized in this way a kind of diagnosing gastric cancer and the detection of prognosis evaluation blood circulation miRNA biomarker
Kit, including nucleotide sequence mir-30c, internal reference；
Further, miR-30c nucleotide sequence: 5 '-TGTAAACATCCTACACTCTCAGC-3 '.
Further, U6 snRNA is as internal reference: the primer sequence of U6 primer upstream and downstream
5 '-CTCGCTTCGGCAGCACA-3 ' and 5 '-AACGCTTCACGAATTTGCGT-3 '.
Further, the calculation method of miR-30c relative expression quantity: 2-ΔCtCycle values calculate, Δ Ct=CtmiR-30c-CtU6。
Another object of the present invention is to provide a kind of diagnosing gastric cancer and prognosis evaluation blood circulation miRNA biomarkers
Detection kit detection method, specifically includes the following steps:
Step 1: the processing of serum sample is taken a blood sample, is centrifuged, is saved backup；
Step 2: the extraction of total serum IgE, the total serum IgE extracted are put in -80 DEG C of refrigerators and save backup；
Step 3: absorbance detection is carried out, the quality of RNA is judged using RNA denaturing formaldehyde gel electrophoresis；
Step 4: carrying out reverse transcription and obtain cDNA, and obtained cDNA is carried out fluorogenic quantitative detection；
Step 5: testing result is analyzed using relative quantification method.
Further, in step 1, the processing of serum sample specifically:
It is all to be included in crowd and take early morning limosis vein blood 3ml, and 8min is centrifuged with 2000rpm with horizontal centrifuge immediately
Serum is separated, the supernatant of suction is placed in centrifuge tube, and 14000x rpm is centrifuged 10min under the conditions of 4 DEG C, and the supernatant of suction is set
In new centrifuge tube, -80 DEG C of refrigerator cryo-conservations are put into for use in the extraction of total serum IgE.
Further, in step 2, the extraction of total serum IgE, specific steps are as follows:
(1) total serum IgE is extracted；
(2) cracking/combination buffer (1ml cracking/combination buffer/0.1g tissue) homogenizer that 10 times of volumes are added is thorough
Bottom mixes；
(3) the homogenate additive of 1/10 volume is added, is vortexed and mixes, places 10 minutes on ice；The above operation is in ice
Upper progress；
(4) acidity-phenol with lysate (disregarding homogenate additive) same volume: chloroform is added) (300ul is cracked
Liquid/300ul acidity-phenol: chloroform), vortex 30-60 seconds, room temperature 10,000g was centrifuged 5 minutes, and split-phase is bad, is centrifuged again；It takes
Supernatant is set in a new pipe, remembers volume；
(5) 1.25 times of 100% ethyl alcohol of volume are added, vortex is mixed, and crosses purification column repeatedly, and volume is no more than 700ul, and 10,
000g is centrifuged about 15 seconds；
(6) 350ul washing lotion 1 is added, is centrifuged 5-10 seconds, cleaning purifying is leant on, and filtered fluid is abandoned in 10,000g centrifugations about 15 seconds；
(7) 10 μ l and Buffer RDD70 μ l of DNase I is added on film (QIAGEN#79254), and 20-30 DEG C is placed 15 points
Clock；
(8) 350ul washing lotion 1 is added, is centrifuged 5-10 seconds, cleaning purifying is leant on, and filtered fluid is abandoned in 10,000g centrifugations 15 seconds；
(9) addition 500ul, washing lotion, 2/3, it is centrifuged 5-10, the second, cleaning purification column is secondary, and 10,000g centrifugations 15 seconds were abandoned
Filtrate is centrifuged 1 minute；
(10) centrifugal column is placed into new collecting pipe, the cleaning agent Elution of 100 μ L95 DEG C preheating is added in column center
In Solution or water without RNA enzyme, room temperature maximum speed is centrifuged 20-30 second, and liquid is the total serum IgE of extraction in collecting pipe,
It is placed on -80 DEG C of preservations.
Further, in step 3, absorbency detection method:
(1) 98ulRNase-free dd H2O is added in the RNA for taking 2ul to dissolve；
(2) with the yield of nucleic acids instrument detection RNA；
(3) absorbance of the measurement at 260mm, 1OD=40ug/ml are calculated as follows:
The concentration of total serum IgE are as follows: A260 × 40 × extension rate.
Further, in step 3, the quality of RNA, specific steps are judged using RNA denaturing formaldehyde gel electrophoresis are as follows:
The preparation of (1) 2.5% agarose gel plate
By agarose powder 2.5g be added 0.5 × TBE100ml in micro-wave oven thermosol until powdered agarose it is completely molten
Solution；When being cooled to 60 DEG C -70 DEG C, 10mg/ml ethidium bromide 5ul is added, pours into the gel slot sealed, thickness about 3-5mm, insertion
Dentation comb；Dentation comb is carefully extracted after molding to be cooled, is put into Agarose horizontal electrophoresis tank, 0.5xTBE flooded glue surface 1cm
Until；
(2) total serum IgE of 0.3 μ g is taken, 5 × sample loading buffer of 1/5 volume is added, 65 DEG C of heating 5min are quenched on ice, with
Eliminate the secondary structure of RNA；The ethidium bromide EtBr, concentration 1.0mg/mL of 0.5-1.0 μ l is added before loading in RNA sample；
The first prerunning 15min in 1 × denaturing formaldehyde gel electrophoresis buffer of the denaturing formaldehyde glue of 1.2% prepared；RNA sample is in 5-
Electrophoresis 30min under the voltage drop of 10V/cm.
Further, in step 4, reverse transcription are as follows:
Defrosting 2 × miRNA RT Reaction Buffer is simultaneously mixed, and miRNA RT Enzyme Mix is put in standby in ice
With addition 2 × miRNA RT Reaction Buffer, 10 μ l, miRNA RT Enzyme in the reaction tube being pre-chilled on ice
It is reacted after 3 μ l to 20 μ l of total volume of 2 μ l of Mix, 5 μ l, RNase-Free ddH2O of total serum IgE, obtained cDNA carries out glimmering
Light quantitative detection.
Further, the real-time fluorescence quantitative PCR reaction of miRNA: according to miRcute Plus miRNA qPCR
Detection kit specification is operated, and 3 secondary orifices of each experimental setup use 7500 real-time fluorescence quantitative PCR instrument of ABI
It is detected.
Further, it in step 5, is analyzed using relative quantification method, miRNA expression quantity is with 2-△CtIt indicates；2-ΔCtCirculation
Value calculates, Δ Ct=(CtMiR-30c experimental group-CtU6 internal reference)。
In conclusion advantages of the present invention and good effect are as follows:
With specificity of the traditional tumour marker in glioma with sensitivity is not high compares, the present invention for
For the tissue of gastric cancer using quantitative fluorescent PCR to patients with gastric cancer, the serum of early carcinoma of stomach and physical examination of healthy population carries out expression quantity detection,
Serum miR-30c is compared in analysis, relative expression quantity meaning that early gastric caacer is diagnosed.For gastric cancer early diagnosis provide it is new
Lesion detection marker.
The present invention and the efficiency of traditional tumour marker CEA, CA199, CA724 in diagnosis are assessed, and find miR-
The diagnostic of 30c is better than traditional tumour marker, and miR-30c low expression indicates patient's prognosis mala.The detection of miR-30c is suitable
Cooperation be gastric cancer diagnosis marker, and be diagnosing gastric cancer develop be new lesion detection marker diagnostic kit, be used for
Therapeutic evaluation and Index for diagnosis, have a good application prospect.
Detailed description of the invention
Fig. 1 is diagnosing gastric cancer and prognosis evaluation blood circulation miRNA biomarker detection provided in an embodiment of the present invention
Kit test method schematic diagram.
Fig. 2 is expression of the circulating tumor marker miR-30c provided in an embodiment of the present invention in gastric cancer group and healthy group
Measure schematic diagram.
Fig. 3 is circulating tumor marker miR-30c provided in an embodiment of the present invention and traditional tumour marker CA72-4,
The curve synoptic diagram of CA19-9 and CEA.
Fig. 4 is Kaplan-Meier survivorship curve schematic diagram provided in an embodiment of the present invention.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Diagnosing gastric cancer and prognosis evaluation blood circulation miRNA biomarker detection reagent provided in an embodiment of the present invention
Box, including nucleotide sequence mir-30c, internal reference；
MiR-30c nucleotide sequence provided in an embodiment of the present invention:
5′-TGTAAACATCCTACACTCTCAGC-3′。
U6snRNA provided in an embodiment of the present invention is as internal reference: the primer sequence of U6 primer upstream and downstream
5 '-CTCGCTTCGGCAGCACA-3 ' and 5 '-AACGCTTCACGAATTTGCGT-3 '.
The calculation method of miR-30c relative expression quantity provided in an embodiment of the present invention: 2-ΔCtCycle values calculate, Δ Ct=
CtmiR-30c-CtU6。
Application principle of the invention is further described with reference to the accompanying drawing；
As shown in Figure 1, diagnosing gastric cancer provided in an embodiment of the present invention and prognosis evaluation blood circulation miRNA biomarker
Detection kit detection method, specifically includes the following steps:
S101: the processing of serum sample is taken a blood sample, is centrifuged, is saved backup；
S102: the extraction of total serum IgE, the total serum IgE extracted are put in -80 DEG C of refrigerators and save backup；
S103: absorbance detection is carried out, the quality of RNA is judged using RNA denaturing formaldehyde gel electrophoresis；
S104: carrying out reverse transcription and obtain cDNA, and obtained cDNA is carried out fluorogenic quantitative detection；
S105: testing result is analyzed using relative quantification method.
In step S101, the processing of serum sample provided in an embodiment of the present invention specifically:
It is all to be included in crowd and take early morning limosis vein blood 3ml, and 8min is centrifuged with 2000rpm with horizontal centrifuge immediately
Serum is separated, the supernatant of suction is placed in centrifuge tube, and 14000x rpm is centrifuged 10min under the conditions of 4 DEG C, and the supernatant of suction is set
In new centrifuge tube, -80 DEG C of refrigerator cryo-conservations are put into for use in the extraction of total serum IgE.
In step S102, the extraction of total serum IgE provided in an embodiment of the present invention, specific steps are as follows:
(1) total serum IgE is extracted；
(2) cracking/combination buffer (1ml cracking/combination buffer/0.1g tissue) homogenizer that 10 times of volumes are added is thorough
Bottom mixes；
(3) the homogenate additive of 1/10 volume is added, is vortexed and mixes, places 10 minutes on ice；The above operation is in ice
Upper progress；
(4) acidity-phenol with lysate (disregarding homogenate additive) same volume: chloroform is added) (300ul is cracked
Liquid/300ul acidity-phenol: chloroform), vortex 30-60 seconds, room temperature 10,000g was centrifuged 5 minutes, and split-phase is bad, is centrifuged again；It takes
Supernatant is set in a new pipe, remembers volume；
(5) 1.25 times of 100% ethyl alcohol of volume are added, vortex is mixed, and crosses purification column repeatedly, and volume is no more than 700ul, and 10,
000g is centrifuged about 15 seconds；
(6) 350ul washing lotion 1 is added, is centrifuged 5-10 seconds, cleaning purifying is leant on, and filtered fluid is abandoned in 10,000g centrifugations about 15 seconds；
(7) 10 μ l and Buffer RDD70 μ l of DNase I is added on film (QIAGEN#79254), and 20-30 DEG C is placed 15 points
Clock；
(8) 350ul washing lotion 1 is added, is centrifuged 5-10 seconds, cleaning purifying is leant on, and filtered fluid is abandoned in 10,000g centrifugations 15 seconds；
(9) addition 500ul, washing lotion, 2/3, it is centrifuged 5-10, the second, cleaning purification column is secondary, and 10,000g centrifugations 15 seconds were abandoned
Filtrate is centrifuged 1 minute；
(10) centrifugal column is placed into new collecting pipe, the cleaning agent Elution of 100 μ L95 DEG C preheating is added in column center
In Solution or water without RNA enzyme, room temperature maximum speed is centrifuged 20-30 second, and liquid is the total serum IgE of extraction in collecting pipe,
It is placed on -80 DEG C of preservations.
In step S103, absorbency detection method provided in an embodiment of the present invention:
(1) 98ulRNase-free dd H2O is added in the RNA for taking 2ul to dissolve；
(2) with the yield of nucleic acids instrument detection RNA；
(3) absorbance of the measurement at 260mm, 1OD=40ug/ml are calculated as follows:
The concentration of total serum IgE are as follows: A260 × 40 × extension rate.
In step S103, the quality provided in an embodiment of the present invention that RNA is judged using RNA denaturing formaldehyde gel electrophoresis, specifically
Step are as follows:
The preparation of (1) 2.5% agarose gel plate
By agarose powder 2.5g be added 0.5 × TBE100ml in micro-wave oven thermosol until powdered agarose it is completely molten
Solution；When being cooled to 60 DEG C -70 DEG C, 10mg/ml ethidium bromide 5ul is added, pours into the gel slot sealed, thickness about 3-5mm, insertion
Dentation comb；Dentation comb is carefully extracted after molding to be cooled, is put into Agarose horizontal electrophoresis tank, 0.5xTBE flooded glue surface 1cm
Until；
(2) total serum IgE of 0.3 μ g is taken, 5 × sample loading buffer of 1/5 volume is added, 65 DEG C of heating 5min are quenched on ice, with
Eliminate the secondary structure of RNA；The ethidium bromide EtBr, concentration 1.0mg/mL of 0.5-1.0 μ l is added before loading in RNA sample；
The first prerunning 15min in 1 × denaturing formaldehyde gel electrophoresis buffer of the denaturing formaldehyde glue of 1.2% prepared；RNA sample is in 5-
Electrophoresis 30min under the voltage drop of 10V/cm.
In step S104, reverse transcription provided in an embodiment of the present invention are as follows:
Defrosting 2 × miRNA RT Reaction Buffer is simultaneously mixed, and miRNA RT Enzyme Mix is put in standby in ice
With addition 2 × miRNA RT Reaction Buffer, 10 μ l, miRNA RT Enzyme in the reaction tube being pre-chilled on ice
It is reacted after 3 μ l to 20 μ l of total volume of 2 μ l of Mix, 5 μ l, RNase-Free ddH2O of total serum IgE, obtained cDNA carries out glimmering
Light quantitative detection.
The real-time fluorescence quantitative PCR of miRNA provided in an embodiment of the present invention reacts: according to miRcute Plus miRNA
QPCR Detection kit specification is operated, and 3 secondary orifices of each experimental setup use 7500 real time fluorescent quantitative of ABI
PCR instrument is detected.
In step S105, in step 5 provided in an embodiment of the present invention, analyzed using relative quantification method, miRNA table
Up to amount with 2-△CtIt indicates；2-ΔCtCycle values calculate, Δ Ct=(CtMiR-30c experimental group-CtU6 internal reference)。
Application principle of the invention is only further described combined with specific embodiments below；
Embodiment 1；
Gastric cancer detection kit
(1) nucleotide sequence mir-30c, specific miR-30c nucleotide sequence:
5′-TGTAAACATCCTACACTCTCAGC-3′。
(2) U6 snRNA is as internal reference: the primer sequence of U6 primer upstream and downstream are as follows:
5 '-CTCGCTTCGGCAGCACA-3 ',
5’-AACGCTTCACGAATTTGCGT-3’。
(3) calculation method of miR-30c relative expression quantity: 2-ΔCtCycle values calculate, Δ Ct=CtmiR-30c-CtU6
One, instrument
Key instrument is as follows
Two, kit detecting step
1. kit detects:
The processing of 1.1 serum samples is all to be included in crowd and takes early morning limosis vein blood 3ml, and uses horizontal centrifuge immediately
Serum is separated with 2000rpm centrifugation 8min, the supernatant of suction is placed in centrifuge tube, and 14000x rpm is centrifuged under the conditions of 4 DEG C
10min, the supernatant of suction are set in new centrifuge tube, are put into -80 DEG C of refrigerator cryo-conservations for use in the extraction of total serum IgE.Always
The extraction of RNA: it is operated, is extracted by miRcuteSerum/Plasma miRNA isolation kit specification
Total serum IgE is put in -80 DEG C of refrigerators and saves backup.
1.2 Total RNAs extractions use miRcute Serum/Plasma miRNA separating kit, reverse transcription and cDNA amplification
Using miRcute Plus miRNA First-Strand cDNA Synthesis kit and miRcute Plus miRNA
QPCR Detection kit (Beijing Tiangeng company), primer, miRNA standard items are synthesized by Beijing Tiangeng biotech firm, are adopted
It is detected with 7500 real-time fluorescence quantitative PCR instrument of ABI.
The ingredient of Beijing Tiangeng company extracts kit
1.3 detailed process
(1) total serum IgE is extracted.
(2) cracking/combination buffer (1ml cracking/combination buffer/0.1g tissue) homogenizer that 10 times of volumes are added is thorough
Bottom mixes.
(3) the homogenate additive of 1/10 volume is added, is vortexed and mixes, places 10 minutes on ice.The above operation is in ice
Upper progress.
(4) acidity-phenol with lysate (disregarding homogenate additive) same volume: chloroform is added) (300ul is cracked
Liquid/300ul acidity-phenol: chloroform), vortex 30-60 seconds, room temperature 10,000g was centrifuged 5 minutes, and split-phase is bad, is centrifuged again.It takes
Supernatant is set in a new pipe, remembers volume.Note: the upper strata aqueous phase in pipe being carefully transferred to new pipe when recycling water phase supernatant
In, following oily phase is not mixed never.
(5) 1.25 times of 100% ethyl alcohol of volume are added, vortex is mixed, and crosses purification column repeatedly, and volume is no more than 700ul, and 10,
000g is centrifuged about 15 seconds.
(6) 350ul washing lotion 1 is added, is centrifuged 5-10 seconds, cleaning purifying is leant on, and filtered fluid is abandoned in 10,000g centrifugations about 15 seconds.
(7) 10 μ l and Buffer RDD70 μ l of DNase I is added on film (QIAGEN#79254), and 20-30 DEG C is placed 15 points
Clock.
(8) 350ul washing lotion 1 is added, is centrifuged 5-10 seconds, cleaning purifying is leant on, and filtered fluid is abandoned in 10,000g centrifugations 15 seconds.
(9) addition 500ul, washing lotion, 2/3, it is centrifuged 5-10, the second, cleaning purification column is secondary, and 10,000g centrifugations 15 seconds were abandoned
Filtrate is centrifuged 1 minute.
(10) centrifugal column is placed into new collecting pipe, the cleaning agent of 95 DEG C of 100 μ L preheatings is added in column center
In Elution Solution or water without RNA enzyme, room temperature maximum speed is centrifuged 20-30 second, and liquid is extraction in collecting pipe
Total serum IgE, can be placed in -80 DEG C of preservations.
1.4 absorbance detection
(1) RNA for taking 2ul to dissolve is added 98ulRNase-free dd H2O, is examined with nucleic acids instrument
The yield of RNA, the absorbance at 260mm are surveyed, the concentration of total serum IgE is calculated as follows in 1OD=40ug/ml
Are as follows: A260 × 40 × extension rate.
(2) according to the light absorption value at 260mm and 280mm, the purity of RNA, the OD260/OD280 ratio of pure rna are detected
It should be close to 2.0 (ratio be preferably between 1.8 and 2.1)；If OD260/OD280 ratio less than 1.8, shows that protein impurities are more；
If OD260/OD280 ratio is greater than 2.2, then show that RNA has degraded.
1.5 RNA denaturing formaldehyde gel electrophoresis
After the total serum IgE for extracting sample, the quality of RNA is generally judged according to the gel electrophoresis figure of RNA.Since RNA is easy
Secondary structure is formed, therefore commonly uses denaturing formaldehyde glue to carry out RNA electrophoresis, obtained electrophoretogram can really reflect the quality of RNA
Situation.
1.6 denaturing formaldehyde glue preparation of reagents
(1) preparation of DEPC water: ddH is measured with graduated cylinder22mlDEPC to 2 liters of ddH is added in draught cupboard in O2L2In O,
Final concentration of 0.1% DEPC.It closes the lid rapidly, mixes, be then placed on middling speed in shaking table and sway at least 4hr, then high pressure is gone out
Bacterium.Bottle cap is unclamped when sterilizing, 15 pounds of sterilizing 20min.
(2) 75% ethyl alcohol is prepared: measuring dehydrated alcohol 75ml with 100ml graduated cylinder, the DEPC water to sterilize is added to be settled to
100ml is fitted into RNA special agent bottle, label, and 4 DEG C of refrigerators save.
The preparation of (3) 50 × TAE: Tris242g, Na need to be weighed by preparing 1 liter of 50 times of TAE buffer2EDTA·2H2O37.2g
Then the ddH of 800ml is added2Dissolution is sufficiently stirred in O, and the acetic acid of 57.1ml is added, mixes well, adds ddH2O is settled to 1L,
Room temperature preservation.Need using when, be diluted to 1 × TAE, such as: need 1 × TAE using 200ml, then measure 4ml 50 ×
The ddH of TAE, 196ml2O, mixing.
(4) 10 × FABuffer (MOPs of formaldehyde agarose buffer:200mM, the NaAc of 50mM,
The EDTA of 10mM): 500ml is prepared, 3.4gNaAC3H2O is weighed and is put into 1000ml beaker, it is processed that 400mlDEPC is added
Deionized water, be added stirrer, be placed on magnetic stirring apparatus and dissolve.Then 20.9gMOPs is added, is placed on magnetic stirring apparatus
Dissolution.Again plus 1.86gNa2EDTA·2H2O is placed on magnetic stirring apparatus and dissolves.(about with 1M sterilized NaOH tune PH to 7.0
With NaOH40ml), it is settled to 500ml with the processed deionized water of DEPC, is fitted into brown bottle, finishes writing label, room temperature is protected from light
It saves.
(5) 5 × denaturing formaldehyde glue sample loading buffers (5 × loading buffer): 10ml is prepared.It prepares in advance
Water saturated bromophenol blue solution: being added about 0.1mg bromophenol blue in 1.5ml centrifuge tube, and it is water-soluble that 1mlDEPC is added
Solution, abundant oscillation dissolution, centrifugation, it is seen that centrifugation bottom of the tube has bromophenol blue powder residue, supernatant liquid, that is, water saturated bromophenol blue
Liquid.In 15ml sterile centrifugation tube, following various composition is sequentially added:
It mixes, packing, -20 DEG C of preservations are common to put 4 DEG C of preservations.
(6) 1 × denaturing formaldehyde gel electrophoresis buffers (1 × running buffer): 20ml10 × FA gel buffer,
The formaldehyde of 4.0ml37%, 176ml water, is put into electrophoresis tank and uses, and generally needs to change this running buffer after 3 electrophoresis
Liquid.
(7) 1.2% denaturing formaldehyde glue: claiming 0.4 gram of agarose, and 10 × FA of 3.34ml gel buffer is added, and is added
30mlDEPC water, micro-wave oven melt, and visually observe grainless suspended matter.It is cooled to about 50-60 DEG C, adds 600 μ l first
Aldehyde pours into the gel mold of 7.5 × 5.0cm.It is inserted into the comb of appropriate length and width, after being placed at room temperature for about 30min
It uses.
1.7 experimental method
The preparation of (1) 2.5% agarose gel plate
By agarose powder 2.5g be added 0.5 × TBE100ml in micro-wave oven thermosol until powdered agarose it is completely molten
Solution.When being cooled to 60 DEG C -70 DEG C, 10mg/ml ethidium bromide 5ul is added, pours into the gel slot sealed, thickness about 3-5mm, insertion
Dentation comb.Dentation comb is carefully extracted after molding to be cooled, is put into Agarose horizontal electrophoresis tank, 0.5xTBE flooded glue surface 1cm
Until.
(2) the general total serum IgE for taking 0.3 μ g, 5 × sample loading buffer of 1/5 volume of addition, 65 DEG C of heating 5min, on ice suddenly
It is cold, to eliminate the secondary structure of RNA.It is recommended that ethidium bromide (EtBr, the concentration of 0.5-1.0 μ l is added before loading in RNA sample
1.0mg/mL), without adding EtBr in glue, the background after such electrophoresis is lower.The denaturing formaldehyde glue of 1.2% prepared is first 1
Prerunning 15min in × denaturing formaldehyde gel electrophoresis buffer.RNA sample electrophoresis 30min under the voltage drop of 5-10V/cm.
Note: chip of expression spectrum RNA sample presentation requirement
(1) RNA total amount is greater than 4ug.
(2) RNA concentration is greater than 50ng/ul, and concentration does not have the upper limit.
(3) RNA volume is greater than 30ul.
(4) self-test before sample presentation determines 260/280 ratio of absorbance of RNA between 1.8 and 2.1；Agarose electrophoresis has two
Item becomes clear band, and upper and lower band brightness ratio is in 2:1 or so.Ratio indicates that the purity of RNA is preferable within the limits prescribed, two
Bright band illustrates that RNA integrality is preferable.
1.8 reverse transcriptions: defrosting 2 × miRNA RT Reaction Buffer is simultaneously mixed, and miRNA RT Enzyme Mix is put
It is spare in ice, 2 × miRNA RT Reaction Buffer, 10 μ l, miRNA RT are added in the reaction tube being pre-chilled on ice
It is reacted, is obtained after 3 μ l to 20 μ l of total volume of 2 μ l of Enzyme Mix, 5 μ l, RNase-Free ddH2O of total serum IgE
CDNA carries out fluorogenic quantitative detection, and all experimental implementations carry out on ice.The real-time fluorescence quantitative PCR of miRNA reacts: according to
MiRcute Plus miRNA qPCR Detection kit specification is operated, 3 secondary orifices of each experimental setup, is used
7500 real-time fluorescence quantitative PCR instrument of ABI is detected.
1.9 calculation method
Experimental results are analyzed using relative quantification method, and miRNA expression quantity is with 2-△CtIt indicates.2-ΔCtCycle values meter
It calculates,
Δ Ct=(CtMiR-30c experimental group-CtU6 internal reference)。
Embodiment 2；
2.1 sample collections:
Autonomous region's expansion clinical sample research in inner mongolia.Be collected into the first affiliated hospital, Medical Colleges of the Inner Mongol from
Total 240 of in January, 2006 in October, 2011, thd patients with gastric carcinoma samples, the gastric cancer tissue sample removed were immediately placed in hospital
It freezes, is then stored in -80 DEG C of refrigerator in liquid nitrogen.The above operation excision sample standard deviation is by two senior Pathology Doctors 's
It is independently diagnosed as gastric cancer, collects pathological data after pathology detection.Tumor invasive depth is according to UICC (International Union Against Cancer
Standard) classification[19], lymphatic metastasis situation, differentiation degree is according to WHO (World Health Organization) Standard Judgement[20], knub position
Pathological tissue of being subject to is reported.
Gender is selected, the non-cancer sample that age and gastric cancer match is derived from Neimenggu Medicine Institute Affiliated Hospital's gastroscope central door
It diagnoses a disease example, and by upper digestion but the detection of gastroscope, pathological biopsy are diagnosed as chronic superficial gastritis or atrophic stomach
Except inflammation, gastric cancer or recurrent tumor patient.Amount to 162, wherein average age be (62.12 ± 12.06) year, age model
Enclose (24-82) year everyone take 5 milliliters of anticoagulant venous blood of EDTA potassium, it is spare that sample is stored in -20 DEG C of refrigerators.Helicobacter pylori
Hpylori Infection Status can be determined as Helicobacter pylori infection by urea breath test.The clinical letter of case-control sample
Breath is obtained by consulting the medical record of the first affiliated hospital, Medical Colleges of the Inner Mongol.All participants have signed by Medical Colleges of the Inner Mongol
The scientific research informed consent form that Ethics Committee formulates.
2.2 RNA extraction steps are same as above
(1) total serum IgE is extracted.
(2) cracking/combination buffer (1ml cracking/combination buffer/0.1g tissue) homogenizer that 10 times of volumes are added is thorough
Bottom mixes.
(3) the homogenate additive of 1/10 volume is added, is vortexed and mixes, places 10 minutes on ice.The above operation is in ice
Upper progress.
(4) acidity-phenol with lysate (disregarding homogenate additive) same volume: chloroform is added) (300ul is cracked
Liquid/300ul acidity-phenol: chloroform), vortex 30-60 seconds, room temperature 10,000g was centrifuged 5 minutes, and split-phase is bad, is centrifuged again.It takes
Supernatant is set in a new pipe, remembers volume.Note: the upper strata aqueous phase in pipe being carefully transferred to new pipe when recycling water phase supernatant
In, following oily phase is not mixed never.
(5) 1.25 times of 100% ethyl alcohol of volume are added, vortex is mixed, and crosses purification column repeatedly, and volume is no more than 700ul, and 10,
000g is centrifuged about 15 seconds.
(6) 350ul washing lotion 1 is added, is centrifuged 5-10 seconds, cleaning purifying is leant on, and filtered fluid is abandoned in 10,000g centrifugations about 15 seconds.
(7) 10 μ l and Buffer RDD70 μ l of DNase I is added on film (QIAGEN#79254), and 20-30 DEG C is placed 15 points
Clock.
(8) 350ul washing lotion 1 is added, is centrifuged 5-10 seconds, cleaning purifying is leant on, and filtered fluid is abandoned in 10,000g centrifugations 15 seconds.
(9) addition 500ul, washing lotion, 2/3, it is centrifuged 5-10, the second, cleaning purification column is secondary, and 10,000g centrifugations 15 seconds were abandoned
Filtrate is centrifuged 1 minute.
(10) centrifugal column is placed into new collecting pipe, the cleaning agent of 95 DEG C of 100 μ L preheatings is added in column center
In Elution Solution or water without RNA enzyme, room temperature maximum speed is centrifuged 20-30 second, and liquid is extraction in collecting pipe
Total serum IgE, can be placed in -80 DEG C of preservations.
2.3 absorbance detection
(1) 98ulRNase-free dd H2O is added in the RNA for taking 2ul to dissolve, with the production of nucleic acids instrument detection RNA
The concentration of total serum IgE is calculated as follows in amount, the absorbance at 260mm, 1OD=40ug/ml are as follows: A260 × 40 × dilution
Multiple.
(2) according to the light absorption value at 260mm and 280mm, the purity of RNA, the OD260/OD280 ratio of pure rna are detected
It should be close to 2.0 (ratio be preferably between 1.8 and 2.1)；If OD260/OD280 ratio less than 1.8, shows that protein impurities are more；
If OD260/OD280 ratio is greater than 2.2, then show that RNA has degraded.
The detection of 2.4 change of serum C A724, CA199 and CEA
Using the dense of the detection of Roche Cobas E601 (Roche) electrochemical luminescence instrument change of serum C A724, CA199 and CEA
The kit of degree, CA724, CA199 and CEA provide (Roche) by Roche Holding Ag.It requires to be detected according to specification,
The detection threshold value (cut off values) of CA724, CEA and CA199 are respectively 6.9ng/ml, 6.5ng/ml, and 27ng/ml.
2.5 statistical method
Data are analyzed using SPSS13.0 statistical software, measurement data usesIt indicates, compares between two groups
Compared with independent samples t test is used, more comparison among groups are compared using single factor test variance.Using miR-30c and CA-724, CEA,
CA199 serum levels draw Receiver Operating Characteristics' (receiver operating characteristic, ROC) curve and
Area under the curve (area under ROC curve, AUC) analyzes the diagnostic value of miR-30c detection, is with P < 0.05
Difference is statistically significant.
2, result:
As shown in Fig. 2, circulating tumor marker miR-30c provided in an embodiment of the present invention is in gastric cancer group and healthy group
Expression quantity schematic diagram.
As shown in Fig. 2, expression quantity not identical (0.41 of the circulating tumor marker miR-30c in gastric cancer group and healthy group
± 0.13vs.0.59 ± 0.12, t=6.7, P < 0.01).
1 circulating tumor marker miR-30c expression quantity of table and clinical pathology move the relationship of speed
Abbreviation:UICC:Union for International Cancer Control, TNM:tumor
node metastasis.
As seen in Table 2, tumor markers CA-724, CA-199 and CEA is commonly used to pass through with circulating tumor marker miR-30c
The diagnosis effect of sensitivity, specificity, efficiency discovery miR-30C of the cutoff value data target assessment in diagnosis is best, sensitive
Degree 80.0%, specificity 89.3%, cutoff value 0.497.Better than traditional tumour marker.
Table 2 CA-724, CA-199 and CEA and circulating tumor marker miR-30c pass through sensitivity, specificity, cutoff value
Data target assesses the Efficacy Results in diagnosis.
SE,standarderror；CI,confidence interval,ROC,receiver-operating
characteristic
As shown in figure 3, circulating tumor marker miR-30c provided in an embodiment of the present invention and traditional tumour marker
The curve synoptic diagram of CA72-4, CA19-9 and CEA.
As shown in figure 3, the song of circulating tumor marker miR-30c and traditional tumour marker CA72-4, CA19-9 and CEA
Area (The area under curves, AUCs) finds that miR-30c, CA 72-4, CA 19-9 and CEA are respectively under line
0.898 (95%CI:0.829-0.967), 0.756 (95%CI:0.639-0.872), 0.641 (95%CI:0.536-0.724)
With 0.662 (95%CI:0.544-0.780), miR-30c area under the curve maximum.
As shown in figure 4, Kaplan-Meier survivorship curve schematic diagram provided in an embodiment of the present invention.
As shown in figure 4, curve shows miR-30c low expression (n=17) and high expression (n=28) prompt patient's prognosis not
Together, ground expresser prognosis mala.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>attached the People's Hospital, Inner Mongolia Medical University (Inner Mongolia Autonomous Region tumour hospital)
<120>diagnosing gastric cancer and prognosis evaluation blood circulation miRNA biomarker detection kit
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tgtaaacatc ctacactctc agc 23
<210> 2
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ctcgcttcgg cagcaca 17
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
aacgcttcac gaatttgcgt 20
Claims (10)
1. a kind of diagnosing gastric cancer and prognosis evaluation blood circulation miRNA biomarker detection kit, which is characterized in that described
Diagnosing gastric cancer and prognosis evaluation blood circulation miRNA biomarker detection kit, including nucleotide sequence mir-30c and interior
Ginseng；
The miR-30c nucleotide sequence are as follows: 5 '-TGTAAACATCCTACACTCTCAGC-3 '；
The internal reference is the primer sequence 5 '-CTCGCTTCGGCAGCACA-3 ' and 5 '-of U6 snRNA:U6 primer upstream and downstream
AACGCTTCACGAATTTGCGT-3’。
2. diagnosing gastric cancer as described in claim 1 and prognosis evaluation blood circulation miRNA biomarker detection kit,
It is characterized in that, the calculation method of the miR-30c relative expression quantity: 2-ΔCtCycle values calculate, Δ Ct=CtmiR-30c-CtU6。
3. a kind of utilize diagnosing gastric cancer and prognosis evaluation blood circulation miRNA biomarker detection reagent described in claim 1
The detection method of box, which is characterized in that the diagnosing gastric cancer and the detection examination of prognosis evaluation blood circulation miRNA biomarker
Detection method includes the following steps for agent box:
Step 1, the processing of serum sample are taken a blood sample, are centrifuged, are saved backup；
Step 2, the extraction of total serum IgE, the total serum IgE extracted are put in -80 DEG C of refrigerators and save backup；
Step 3 is carried out absorbance detection, the quality of RNA is judged using RNA denaturing formaldehyde gel electrophoresis；
Step 4, carries out reverse transcription and obtains cDNA, and obtained cDNA is carried out fluorogenic quantitative detection；
Step 5 analyzes testing result using relative quantification method.
4. diagnosing gastric cancer as claimed in claim 3 and prognosis evaluation blood circulation miRNA biomarker detection kit
Detection method, which is characterized in that in the step 1, the processing of serum sample specifically:
It is all to be included in crowd and take early morning limosis vein blood 3ml, and immediately with horizontal centrifuge with 2000rpm centrifugation 8min separation
The supernatant of serum, suction is placed in centrifuge tube, and 14000x rpm is centrifuged 10min under the conditions of 4 DEG C, and the supernatant of suction is set new
In centrifuge tube, -80 DEG C of refrigerator cryo-conservations are put into for use in the extraction of total serum IgE.
5. diagnosing gastric cancer as claimed in claim 3 and prognosis evaluation blood circulation miRNA biomarker detection kit
Detection method, which is characterized in that in the step 2, the extraction of total serum IgE, specific steps are as follows:
(1) total serum IgE is extracted；
(2) cracking/combination buffer homogenizer that 10 times of volumes are added thoroughly mixes；
(3) the homogenate additive of 1/10 volume is added, is vortexed and mixes, places 10 minutes on ice；The above operation on ice into
Row；
(4) acidity-phenol with lysate same volume: chloroform is added, 300ul lysate/300ul acidity-phenol: chloroform,
Vortex 30-60 seconds, room temperature 10,000g was centrifuged 5 minutes, and split-phase is bad, is centrifuged again；It takes supernatant to set in a new pipe, remembers volume；
(5) 1.25 times of 100% ethyl alcohol of volume are added, vortex is mixed, and crosses purification column repeatedly, and volume is no more than 700ul, and 10,000g
Centrifugation about 15 seconds；
(6) 350ul washing lotion 1 is added, is centrifuged 5-10 seconds, cleaning purifying is leant on, and filtered fluid is abandoned in 10,000g centrifugations about 15 seconds；
(7) DNaseI10 μ l and Buffer RDD70 μ l is added on film, and 20-30 DEG C is placed 15 minutes；
(8) 350ul washing lotion 1 is added, is centrifuged 5-10 seconds, cleaning purifying is leant on, and filtered fluid is abandoned in 10,000g centrifugations 15 seconds；
(9) addition 500ul, washing lotion, 2/3, it is centrifuged 5-10, the second, cleaning purification column is secondary, and 10,000g centrifugations 15 seconds are abandoned and filtered
Liquid is centrifuged 1 minute；
(10) centrifugal column is placed into new collecting pipe, the cleaning agent Elution of 100 μ L95 DEG C preheating is added in column center
In Solution or water without RNA enzyme, room temperature maximum speed is centrifuged 20-30 second, and liquid is the total serum IgE of extraction in collecting pipe,
It is placed on -80 DEG C of preservations.
6. diagnosing gastric cancer as claimed in claim 3 and prognosis evaluation blood circulation miRNA biomarker detection kit
Detection method, which is characterized in that in the step 3, absorbency detection method:
(1) 98ulRNase-free dd H is added in the RNA for taking 2ul to dissolve2O；
(2) with the yield of nucleic acids instrument detection RNA；
(3) absorbance of the measurement at 260mm, 1OD=40ug/ml are calculated as follows:
The concentration of total serum IgE are as follows: A260 × 40 × extension rate.
7. diagnosing gastric cancer as claimed in claim 2 and prognosis evaluation blood circulation miRNA biomarker detection kit
Detection method, which is characterized in that in the step 3, the quality of RNA, specific steps are judged using RNA denaturing formaldehyde gel electrophoresis
Are as follows:
The preparation of (1) 2.5% agarose gel plate
By agarose powder 2.5g be added 0.5 × TBE100ml in micro-wave oven thermosol until powdered agarose be completely dissolved；
When being cooled to 60 DEG C -70 DEG C, 10mg/ml ethidium bromide 5ul is added, pours into the gel slot sealed, thickness about 3-5mm, insertion tooth
Shape comb；Dentation comb is carefully extracted after molding to be cooled, is put into Agarose horizontal electrophoresis tank, 0.5xTBE flooded glue surface 1cm and is
Only；
(2) total serum IgE of 0.3 μ g is taken, 5 × sample loading buffer of 1/5 volume is added, 65 DEG C of heating 5min are quenched on ice, to eliminate
The secondary structure of RNA；The ethidium bromide EtBr, concentration 1.0mg/mL of 0.5-1.0 μ l is added before loading in RNA sample；It prepares
The first prerunning 15min in 1 × denaturing formaldehyde gel electrophoresis buffer of 1.2% denaturing formaldehyde glue；RNA sample is in 5-10V/cm
Voltage drop under electrophoresis 30min.
8. diagnosing gastric cancer as claimed in claim 3 and prognosis evaluation blood circulation miRNA biomarker detection kit
Detection method, which is characterized in that in the step 4, reverse transcription are as follows:
Thaw 2 × miRNART ReactionBuffer simultaneously mix, miRNART Enzyme Mix be put in it is spare in ice, on ice
2 × miRNART ReactionBuffer, 10 μ l, 2 μ l of miRNA RT Enzyme Mix are added in the reaction tube of pre-cooling, always
5 μ l, RNase-Free ddH of RNA2It is reacted after 3 μ l of O to 20 μ l of total volume, obtained cDNA carries out fluorescent quantitation inspection
It surveys.
9. diagnosing gastric cancer as claimed in claim 3 and prognosis evaluation blood circulation miRNA biomarker detection kit
Detection method, which is characterized in that the real-time fluorescence quantitative PCR of the miRNA reacts: according to miRcute Plus miRNA
QPCR Detectionkit specification is operated, and 3 secondary orifices of each experimental setup use 7500 real time fluorescent quantitative of ABI
PCR instrument is detected.
10. diagnosing gastric cancer as claimed in claim 3 and prognosis evaluation blood circulation miRNA biomarker detection kit
Detection method, which is characterized in that in the step 5, analyzed using relative quantification method, miRNA expression quantity is with 2-△CtTable
Show；2-ΔCtCycle values calculate, Δ Ct=(CtMiR-30c experimental group-CtU6 internal reference)。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111206094A (en) * | 2018-11-21 | 2020-05-29 | 内蒙古医科大学附属人民医院 | PiRNA biomarker for noninvasive early diagnosis of colorectal cancer and rectal cancer and detection kit |
| CN112133365A (en) * | 2020-09-03 | 2020-12-25 | 南方医科大学南方医院 | Gene set for evaluating tumor microenvironment, scoring model and application of gene set |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160362689A1 (en) * | 2012-08-29 | 2016-12-15 | City Of Hope | Differentially expressed microrna molecules for the treatment and diagnosis of cancer |
| US20170130276A1 (en) * | 2014-06-16 | 2017-05-11 | Toray Industries, Inc. | Stomach cancer detection kit or device, and detection method |
-
2019
- 2019-07-02 CN CN201910588811.0A patent/CN110229907A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160362689A1 (en) * | 2012-08-29 | 2016-12-15 | City Of Hope | Differentially expressed microrna molecules for the treatment and diagnosis of cancer |
| US20170130276A1 (en) * | 2014-06-16 | 2017-05-11 | Toray Industries, Inc. | Stomach cancer detection kit or device, and detection method |
Non-Patent Citations (6)
| Title |
|---|
| JING-MEI CAO等: "MiR-30c-5p suppresses migration, invasion and epithelial to mesenchymal transition of gastric cancer via targeting MTA1", BIOMED PHARMACOTHER, vol. 93, pages 554 - 560, XP085147917, DOI: 10.1016/j.biopha.2017.06.084 * |
| WENYAN HAN等: "Expression of miR-30c and BCL-9 in gastric carcinoma tissues and their function in the development of gastric cancer", ONCOL LETT, vol. 16, no. 2, pages 2416 - 2426 * |
| 杨洁泉等: "微小RNA-30c在胃癌组织中的表达及意义", 中国老年学杂志, vol. 38, no. 18 * |
| 牟永平等: "MicroRNA-30c相对表达量与胃癌预后相关性分析", 中华肿瘤防治杂志, vol. 21, no. 20, pages 1598 - 1601 * |
| 牟永平等: "血清miRNA-30c在胃癌早期诊断中的作用", 肿瘤研究与临床, vol. 30, no. 5 * |
| 霍小蕾等: "miR-30c对胃癌细胞MGC803增殖和侵袭能力的影响", 长治医学院学报, vol. 32, no. 5, pages 331 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111206094A (en) * | 2018-11-21 | 2020-05-29 | 内蒙古医科大学附属人民医院 | PiRNA biomarker for noninvasive early diagnosis of colorectal cancer and rectal cancer and detection kit |
| CN111206094B (en) * | 2018-11-21 | 2024-01-19 | 北京大学肿瘤医院内蒙古医院(内蒙古医科大学附属肿瘤医院、内蒙古自治区肿瘤医院、内蒙古自治区癌症中心) | PiRNA biomarker for noninvasive early diagnosis of colorectal cancer and rectal cancer and detection kit |
| CN112133365A (en) * | 2020-09-03 | 2020-12-25 | 南方医科大学南方医院 | Gene set for evaluating tumor microenvironment, scoring model and application of gene set |
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