CN106636375A - Marker miR-126-3P for HER-2 positive breast cancer tissue, and application and diagnosis kit of marker miR-126-3P - Google Patents
Marker miR-126-3P for HER-2 positive breast cancer tissue, and application and diagnosis kit of marker miR-126-3P Download PDFInfo
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Abstract
The invention discloses a marker miR-126-3P for predicting and diagnosing a HER-2 positive breast cancer tissue, and application and diagnosis kit of the marker. The miR-126-3P can be used as a treatment target for the HER-2 positive breast cancer. The diagnosis kit comprises a nucleic acid probe for the miR-126-3P, a nucleic acid probe of positive contrast Scramble, a nucleic acid probe of positive contrast U6snRNA, an in situ hybridization buffer solution and protease K powder. The breast cancer molecule marker miR-126-3P disclosed by the invention for diagnosing the HER-2 positive breast cancer has the characteristics of simple operation, convenience in material taking, safety, no invasion, high specificity and high accuracy.
Description
Technical field
The present invention relates to field of biomedicine technology, specifically, be related to one kind can be used to indicating and diagnose HER-2 it is positive
The mark miR-126-3p of property breast cancer tissue, the application of the mark and diagnostic kit.
Background technology
Breast cancer is modal malignant tumour in female population in the world, is also the dead most common reason of female cancer
One of.Breast cancer is one group of different substantiality disease, including various histological types, and every kind of histological type is respectively provided with different histology tables
Existing, different biological behaviour, different clinical manifestations, different therapeutic response and treatment results.Perou etc. adopts first base
Because microarray and prognosis Clustering Analysis Technology are measured to breast cancer sample, it is found that different mastocarcinoma gene phenotypes are faced with it
Bed biological behaviour and Prognostic significance are close.According to its heredity and molecular linkage map, will can be infiltrated using chip of expression spectrum technology
Property breast cancer falls into 5 types：Respectively Luminal A types (ER and/or PR+, HER-2-, Ki-67<L4%), Luminal Type Bs
(ER and/or PR+, HER-2-, Ki-67 >=14%；ER and/or any level of PR+, HER-2+, Ki 67), HER-2 overexpression types
(ER-, PR-, HER-2+) and three female (triple-negative breast carcinomas, TNBCs) (ER-, PR-,
HER-2-).Wherein hormone therapy is effective to ER (+) type, and trastuzumab treatment is effective to HER-2 overexpression types.TNBC types
Account for pathogenesis of breast carcinoma sum 15%-20%, and because its hormone receptor site three it is negative, to anti-hormonal therapy and Trastuzumab
Fail to respond to any medical treatment, unique therapeutic scheme is systemic chemotherapy.Therefore the breast cancer of 4 kinds of different immunophenotypes has relatively independent
Biological behaviour and clinical prognosis, study the corresponding pathological characters of each hypotype breast cancer individualized treatment side new for offer
Method, realizes that precisely medical treatment is significant.
MicroRNA (miRNA) is the non-protein coding small molecule RNA of length about 18-25nt, is widely present in various true
In nucleus biological cell.In recent years numerous studies show that miRNAs plays an important role to the generation of human tumor, development,
And play the role of oncogene and tumor suppressor gene.Research in recent years is found that a series of exception tables in breast cancer tissue
The miRNAs for reaching.Wherein miR-126-3p is found in metastatic breast cancer clone notable low expression, and is proved in suppression
Key effect is played in metastases processed.But, breast cancer tissue's evidence is lacked so far and shows miR-126-3p not
Whether there is difference with the breast carcinoma of immunophenotype.
The content of the invention
The technical problem to be solved is the mark miR-126- that can express HER-2 positive breast cancerous tissues
The application and its diagnostic kit of 3p, the mark in the diagnostic reagent for preparing diagnosis HER-2 positive breast cancers.
The application shows that miR-126-3p can be used as the therapy target of specific molecular hypotype breast cancer.
To solve above-mentioned technical problem, the invention provides a kind of diagnostic kit of HER-2 positive breast cancers, including：
The nucleic acid probe of MiR-126-3p, the nucleic acid probe of negative control Scramble, the nucleic acid of positive control U6snRNA
Probe, in situ hybridization buffer solution and Proteinase K powder.
Preferably, the nucleic acid probe sequence of the MiR-126-3p such as SEQ ID NO：Shown in 1.
Preferably, the nucleic acid probe sequence of the negative control Scramble such as SEQ ID NO：Shown in 2.
Preferably, the nucleic acid probe sequence of the positive control U6snRNA such as SEQ ID NO：Shown in 3.
Disclosed herein as well is the mark miR-126-3p of HER-2 positive breast cancerous tissues is positive in preparation diagnosis HER-2
Application in the diagnostic reagent of property breast cancer, it is characterised in that the mark miR-126- of the HER-2 positive breasts cancerous tissue
The nucleic acid probe sequence of 3p such as SEQ ID NO：Shown in 1.
Preferably, the miR-126-3p contents in breast cancer tissue are detected using FISH detection methods.
Preferably, including step：
(1) nucleic acid probe, the nucleic acid probe of negative control Scramble and the positive control of MiR-126-3p are prepared
The nucleic acid probe of U6snRNA；
(2) hybridization step
1. organization chip is put in baking oven, temperature is adjusted to 60-65 degree, dries wax 1 hour；
2. the pretreatment of probe：
Prepare 1 × miRNA-ISH buffer solutions：2 × miRNA-ISH of 10ml buffer solutions and 10ml DEPC H2O, by 40ul
Nucleic acid probe be placed in the PCR pipe without RNase, 90 DEG C of denaturation 4min are placed on ice, add after quick centrifugation 20ml 1 ×
MiRNA-ISH is buffered, and is dispensed by the volume of 1-2ml, and direct freeze thawing when to be used is used；
3. buffer：
10×PBS：40g NaCl, 1g KCl, 7.2g Na2PO4,1.2g KH2PO4 are taken, with HCl pH 7.4 is adjusted, used
DEPC water is settled to 500ml；
1×PBT：50ml 10 × PBS and 0.5ml Tween are taken, with DEPC water 500ml is settled to；
1M Tris-HCl, PH7.4：30.475g Tris alkali, plus 150mlDEPC water, with concentrated hydrochloric acid PH to 7.4 is adjusted, and is used
DEPC water is settled to 250ml；
Proteinase K stores the preparation of liquid：With 10mM Tris-HCl PH.7.5 diluted protein enzyme K to 20mg/ml；
Maleic acid buffer solution：0.1M maleic acids, 0.15MNaCl adjusts PH to 7.5 with NaOH；
NBT/BCIP dilutions：100ml 1M/L Tris-HCl, 20ml 5M/L NaCl, 880ml DEPC ddH2O,
pH9.5；
4. sample dewaxing：
3 each 5min of dimethylbenzene,
Ethanol embathes 10 times about 100%,
Ethanol embathes 10 times about 100%
Ethanol 100%5min,
Ethanol embathes 10 times about 96%,
Ethanol 96%5min,
Ethanol embathes 10 times about 70%,
Ethanol 70%5min,
PBS 2-5min；
5. protease digestion sample：
37 DEG C of Proteinase K 15ug/mL processes 10-30min, PBS 2 times, simply shakes；
6. dehydration of alcohol：
Ethanol embathes 10 times about 70%
Ethanol 70%1min,
Ethanol embathes 10 times about 96%,
Ethanol 96%1min,
Ethanol embathes 10 times about 100%,
Ethanol 100%1min, is placed on clean paper and is air-dried 15min；
7. hybridize：
Add the hybridization solution of 50-100ul on every slide；22mm × 22mm cover glasses are covered on slide, glue mounting is used,
50 DEG C of 1h of hybridization temperature；
8. it is strict to develop a film：
Mounting glue is torn with tweezers are careful, slide are put into following solution gradient and developed a film：
50 DEG C of 5 × SSC 5min hybridization temperatures,
50 DEG C of 1 × SSC 5min hybridization temperatures,
50 DEG C of 0.2 × SSC 5min hybridization temperatures,
0.2 × SSC 5min room temperatures,
Room temperature, 1 time × 5min of PBS；
9. immune detection：
Slide is dried, confining liquid is added dropwise in room temperature wet box, close 15min；Blotted after confining liquid with paper, be placed on wet box
1:800anti-DIG-AP Fab fragments, 4 DEG C overnight；Room temperature, 3 times × 3min of PBST；
10. color reaction and mounting is redyed：
Lucifuge reaction 30 DEG C of colour developings 2h (400ul/ slides) of NBT/BCIP, room temperature, 3 times × 5min of PBST；Wash in water
Piece, 2 1min；Plus 200ul core fast red dye liquors, -1 minute 10 seconds；Slice, thin piece is rinsed 10 minutes in running water, dehydration of alcohol；
Use resinene mounting；
(3) in situ hybridization testing result judges：
By double-blind study independent evaluations, its positive cell number and dye levels, positive cell number scoring are evaluated respectively：Have no
Positive cell is 0, positive cell<5% is 1, and 6%～25% is 2, and 25%～50% is 3, and 51%～75% is 4,>75% is
5；Dye levels score：Have no that dyeing is 0, it is 1 light gray coloured particles occur, it is 2 Dark grey occur, it is 3 black particle occur, always
Appraisal result=staining positive cells number scoring × dye levels scoring.
Compared with prior art, the mark miR-126-3p of HER-2 positive breasts cancerous tissue of the present invention, application
And its diagnostic kit, reach following effect：
There is operation letter using the breast cancer molecular mark miR-126-3p diagnosis HER-2 positive breast cancers of the present invention
It is single, draw materials convenience, safe hurtless measure, high specific, high precision the characteristics of.
Description of the drawings
Accompanying drawing described herein is used for providing a further understanding of the present invention, constitutes the part of the present invention, this
Bright schematic description and description does not constitute inappropriate limitation of the present invention for explaining the present invention.In the accompanying drawings：
Fig. 1 is using the expression ROC curve for disliking type diagnostic good to HER-2 positive breast tumors of miR-126-3p；
Fig. 2 is bent to the ROC of HER-2 positive breast cancers TNM stage and Index for diagnosis using the expression of miR-126-3p
Line.
Specific embodiment
As in specification and claim some vocabulary used in censuring specific components.Those skilled in the art should
It is understood that hardware manufacturer may call same component with different nouns.This specification and claims are not with name
The difference of title is used as distinguishing the mode of component, but the difference with component functionally is used as the criterion distinguished.Such as logical
The "comprising" of piece specification and claim mentioned in is an open language, therefore should be construed to " include but do not limit
In "." substantially " refer in receivable error range, those skilled in the art can solve described in the range of certain error
Technical problem, basically reaches the technique effect.Specification subsequent descriptions are to implement the better embodiment of the present invention, so described
Description be by illustrate the present invention rule for the purpose of, be not limited to the scope of the present invention.Protection scope of the present invention
When being defined depending on the claims person of defining.
The present invention is described in further detail below in conjunction with accompanying drawing, but it is not as a limitation of the invention.
1st, material：The paraffin organization sample for being adopted is from mastosis section of Central Hospital, Huzhou City in January, 2009 to 2015
Year December surgical resection, pathology department the achieve, breast cancer tissue that histopathology data and clinical and pathological data are complete.Its
The Jing pathological diagnosis of Zhong You98Li breast cancer tissues turns out to be infiltration ductal carcinomas of breast, and HER-2 is positive.Collect 98 patients'
Breast cancer tissue and its pairing cancer beside organism (apart from primary lesion 5cm).It is female patient, age 31-74 year, average age
48.98 years old.Radiotherapy, chemotherapy and immunization therapy etc. had not been carried out before corrective surgery, in operation.
2nd, in situ hybridization detects the amplified signal of miR-126-3p
(1) prepared by probe
Nucleic acid probe design is the digoxin double labelling at 5 ' ends and 3 ' ends, is synthesized from Shanghai Sheng Gong companies.
MiR-126-3p probe sequences are：5’-CGCATTATTACTCACGGTACGA-3’；Negative control Scramble's
Sequence is 5 '-GTGTAACACGTCTATACGCCCA-3 '；The sequence of positive control U6snRNA probes be 5 '-
CACGAATTTGCGTGTCATCCTT-3’。
(2) hybridization step
1. organization chip is put in baking oven, temperature is adjusted to 60-65 degree, dries wax 1 hour；
2. the pretreatment of probe：
Prepare 1 × miRNA-ISH buffer solutions：2 × miRNA-ISH of 10ml buffer solutions and 10ml DEPC H2O, by 40ul
Nucleic acid probe be placed in the PCR pipe without RNase, 90 DEG C of denaturation 4min are placed on ice, add after quick centrifugation 20ml 1 ×
MiRNA-ISH is buffered, and is dispensed by the volume of 1-2ml, and direct freeze thawing when to be used is used；
3. buffer：
10×PBS：40g NaCl, 1g KCl, 7.2g Na2PO4,1.2g KH2PO4 are taken, with HCl pH 7.4 is adjusted, used
DEPC water is settled to 500ml；
1×PBT：50ml 10 × PBS and 0.5ml Tween are taken, with DEPC water 500ml is settled to；
1M Tris-HCl, PH7.4：30.475g Tris alkali, plus 150mlDEPC water, with concentrated hydrochloric acid PH to 7.4 is adjusted, and is used
DEPC water is settled to 250ml；
Proteinase K stores the preparation of liquid：With 10mM Tris-HCl PH.7.5 diluted protein enzyme K to 20mg/ml；
Maleic acid buffer solution：0.1M maleic acids, 0.15MNaCl adjusts PH to 7.5 with NaOH；
NBT/BCIP dilutions：100ml 1M/L Tris-HCl, 20ml 5M/L NaCl, 880ml DEPC ddH2O,
pH9.5；
4. sample dewaxing：
3 each 5min of dimethylbenzene,
Ethanol embathes 10 times about 100%,
Ethanol embathes 10 times about 100%
Ethanol 100%5min,
Ethanol embathes 10 times about 96%,
Ethanol 96%5min,
Ethanol embathes 10 times about 70%,
Ethanol 70%5min,
PBS 2-5min；
5. protease digestion sample：
37 DEG C of Proteinase K 15ug/mL processes 10-30min, PBS 2 times, simply shakes；
6. dehydration of alcohol：
Ethanol embathes 10 times about 70%
Ethanol 70%1min,
Ethanol embathes 10 times about 96%,
Ethanol 96%1min,
Ethanol embathes 10 times about 100%,
Ethanol 100%1min, is placed on clean paper and is air-dried 15min；
7. hybridize：
Add the hybridization solution of 50-100ul on every slide；22mm × 22mm cover glasses are covered on slide, glue mounting is used,
50 DEG C of 1h of hybridization temperature；
8. it is strict to develop a film：
Mounting glue is torn with tweezers are careful, slide are put into following solution gradient and developed a film：
50 DEG C of 5 × SSC 5min hybridization temperatures,
50 DEG C of 1 × SSC 5min hybridization temperatures,
50 DEG C of 0.2 × SSC 5min hybridization temperatures,
0.2 × SSC 5min room temperatures,
Room temperature, 1 time × 5min of PBS；
9. immune detection：
Slide is dried, confining liquid is added dropwise in room temperature wet box, close 15min；Blotted after confining liquid with paper, be placed on wet box
1:800anti-DIG-AP Fab fragments, 4 DEG C overnight；Room temperature, 3 times × 3min of PBST；
10. color reaction and mounting is redyed：
Lucifuge reaction 30 DEG C of colour developings 2h (400ul/ slides) of NBT/BCIP, room temperature, 3 times × 5min of PBST；Wash in water
Piece, 2 1min；Plus 200ul core fast red dye liquors, -1 minute 10 seconds；Slice, thin piece is rinsed 10 minutes in running water, dehydration of alcohol；
Use resinene mounting；
(3) in situ hybridization testing result judges：
By double-blind study independent evaluations, its positive cell number and dye levels, positive cell number scoring are evaluated respectively：Have no
Positive cell is 0, positive cell<5% is 1, and 6%～25% is 2, and 25%～50% is 3, and 51%～75% is 4,>75% is
5；Dye levels score：Have no that dyeing is 0, it is 1 light gray coloured particles occur, it is 2 Dark grey occur, it is 3 black particle occur, always
Appraisal result=staining positive cells number scoring × dye levels scoring.
As a result
The expression of results of the miR-126-3p of 98 breast cancer tissues and its pairing cancer beside organism is shown in Table 4.
Expressions of the miR-126-3p in 98 cancerous tissues is 6.02 ± 2.713, in 98 cancer beside organisms that it is matched
In expression be 3.19 ± 1.448, two groups have significant difference (t=9.321, p<0.001), illustrate that miR-126-3p exists
Expression in cancerous tissue is significantly higher than non-cancer tissue, can be used as the evaluation index for judging benign tumors evil.Table 1 is miR-126-
Expressions of the 3p in breast cancer tissue and the correlation between lymphatic metastasis and TNM stage：Drenching is occurring in miR-126-3p
The expression (6.66 ± 2.700) in the breast cancer tissue of transfer is fawned on higher than the breast cancer tissue for lymphatic metastasis do not occur
Difference statistically significant (t=2.442, p=0.016) between (5.35 ± 2.589), and two groups；MiR-126-3p is at TNM point
Phase is the expression (5.44 ± 2.536) in the breast cancer tissue of I-II phases less than the breast cancer group that TNM stage is the III-IV phases
Difference statistically significant (t=2.575, p=0.012) between the expression (6.83 ± 2.774) knitted and two groups.Additionally,
We further have rated miR-126-3p expressions and the correlation between age and tumor size, as a result show, miR-
126-3p expressions are 0.106 (p=0.301) with the coefficient correlation at age, are 0.158 (p=with the coefficient correlation of tumor size
0.120).Result above shows that the expression of miR-126-3p increases in there is the higher tissue of lymphatic metastasis and TNM stage
Plus, it is unrelated with age and tumor size, illustrate that the expression of miR-126-3p is close with the grade malignancy of HER-2 positive breast cancers
Correlation, high expression of the miR-126-3p in HER-2 positive breast cancerous tissues has the clinical effect for clearly pointing out prognosis mala.
Table 1：Expressions of the miR-126-3p in breast cancer tissue and the correlation between lymphatic metastasis and TNM stage
| Parameter | Number of cases | miR-126-3p | T/p values |
| Lymphatic metastasis | |||
| Have | 50 | 6.66±2.700 | T=2.442 |
| Nothing | 48 | 5.35±2.589 | P=0.016 |
| TNM stage | |||
| I-II | 57 | 5.44±2.536 | T=2.575 |
| III-IV | 41 | 6.83±2.774 | P=0.012 |
For the expression for further the evaluating miR-126-3p diagnostic value benign and malignant to tumour and to tumor-infiltrated
The forewarning function of depth, grade malignancy, we have rated respectively miR-126-3p in area using diagnostic characteristic curve (ROC) analysis
The diagnostic value divided in cancerous tissue and cancer beside organism, high TNM stage (III-IV) and low TNM stage (I-II).The base of the method
Present principles are：By the movement for diagnosing boundary's point (cutoff point/cutoff value), multipair sensitivity is obtained
(sensitivity) and misdiagnosis rate (1- specificity specificity), with sensitivity without the longitudinal axis, with misdiagnosis rate as transverse axis, connection
Each point-rendering curve, area (AUC) then under calculated curve, area is bigger, and diagnostic value is higher.Fig. 1 is to be commented with ROC analyses
The expression of the valency miR-126-3p diagnostic value for disliking type good to HER-2 positive breast tumors.
Area is 0.809 under ROC curve, and the standard of area is mistaken for the expression of 0.031, miR-126-3p for judging to swell
Benign and malignant degree tool significance (P=0.000) of knurl, the expression of miR-126-3p is higher, malignancy
It is stronger.The credibility interval of area for (0.748,0.871) include 0.5, draw identical conclusion.According to ROC curve each point institute
Corresponding sensitivity and misdiagnosis rate (1- specificities), are shown in Table 2, select the maximum (0.551) of sensitivity-misdiagnosis rate corresponding
The expression value (4.5) of miR-126-3p is diagnosis circle's point value, i.e. cutoff values.
Table 2 utilizes the seat of the expression of the miR-126-3p ROC curve for disliking type diagnostic good to HER-2 positive breast tumors
Mark
Fig. 2 is to HER-2 positive breast cancer TNM stage degree and pre- with the expression of ROC assay miR-126-3p
The diagnostic value for judging afterwards.
Area is 0.650 under ROC curve, and the standard of area is mistaken for the expression of 0.057, miR-126-3p for judging to dislike
Property tumour infiltration degree tool significance (p=0.011), the expression of miR-126-3p is higher, the infiltration degree of tumour
Higher, TNM stage is higher, and prognosis is poorer.The credibility interval of area for (0.539,0.761) include 0.5, draw identical tie
By.Sensitivity and misdiagnosis rate (1- specificities) according to corresponding to ROC curve each point, is shown in Table 3, selects sensitivity-misdiagnosis rate
The expression value (7.0) of the corresponding miR-126-3p of maximum (0.225) is diagnosis circle's point value, i.e. cutoff values.
Table 3 is using the expression of miR-126-3p to HER-2 positive breast cancers TNM stage and the ROC curve of Index for diagnosis
Coordinate
The fairly large clinical verification experimental data according to more than, diagnosis of the exploitation suitable for HER-2 breast cancer patients with positive
Kit, the kit can detect whether the tumor tissues of patient express excessive miR-126-3p (cutoff values are 4.5), carry
Show that patient can adopt the medicine of corresponding antagonism miR-126-3p be treated, miR-126-3p values are higher, and (cutoff values are
7.0) prompting tumor patient TNM stage is higher, and prognosis is poorer.
The kit includes：MiR-126-3p nucleic acid probes, negative control Scramble nucleic acid probes, positive control U6
SnRNA probes, in situ hybridization buffer solution, Proteinase K powder.
Kit using method is referring to hybridization step.
The expression of results of the miR-126-3p of 4 98 breast cancer tissues of table and its pairing cancer beside organism
Described above illustrates and describes some preferred embodiments of the present invention, but as previously mentioned, it should be understood that the present invention
Be not limited to form disclosed herein, be not to be taken as the exclusion to other embodiment, and can be used for various other combinations,
Modification and environment, and can be in invention contemplated scope described herein, by above-mentioned teaching or the technology or knowledge of association area
It is modified.And change that those skilled in the art are carried out and change be without departing from the spirit and scope of the present invention, then all should be at this
In the protection domain of bright claims.
SEQUENCE LISTING
<110>Central Hospital, Huzhou City
<120>The mark miR-126-3p of HER-2 positive breast cancerous tissues, application and its diagnostic kit
<130> 160033
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence
<400> 1
cgcattatta ctcacggtac ga 22
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence
<400> 2
gtgtaacacg tctatacgcc ca 22
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence
<400> 3
cacgaatttg cgtgtcatcc tt 22
Claims (7)
1. a kind of diagnostic kit of HER-2 positive breast cancers, it is characterised in that include：
The nucleic acid probe of MiR-126-3p, the nucleic acid probe of negative control Scramble, the nucleic acid of positive control U6snRNA are visited
Pin, in situ hybridization buffer solution and Proteinase K powder.
2. the diagnostic kit of HER-2 positive breast cancers according to claim 3, it is characterised in that the MiR-126-
The nucleic acid probe sequence of 3p such as SEQ ID NO：Shown in 1.
3. the diagnostic kit of HER-2 positive breast cancers according to claim 3, it is characterised in that
The nucleic acid probe sequence of the negative control Scramble such as SEQ ID NO：Shown in 2.
4. the diagnostic kit of HER-2 positive breast cancers according to claim 3, it is characterised in that
The nucleic acid probe sequence of the positive control U6snRNA such as SEQ ID NO：Shown in 3.
The mark miR-126-3p of 5.HER-2 positive breast cancerous tissues is preparing the diagnosis examination of diagnosis HER-2 positive breast cancers
Application in agent, it is characterised in that the nucleic acid probe sequence of the mark miR-126-3p of the HER-2 positive breasts cancerous tissue
Such as SEQ ID NO：Shown in 1.
6. the mark miR-126-3p of HER-2 positive breasts cancerous tissue according to claim 5 is preparing diagnosis HER-2
Application in the diagnostic reagent of positive breast cancer, it is characterised in that using in FISH detection methods detection breast cancer tissue
MiR-126-3p contents.
7. the mark miR-126-3p of HER-2 positive breasts cancerous tissue according to claim 5 is preparing diagnosis HER-2
Application in the diagnostic reagent of positive breast cancer, it is characterised in that including step：
(1) nucleic acid probe, the nucleic acid probe of negative control Scramble and the positive control U6snRNA of MiR-126-3p are prepared
Nucleic acid probe；
(2) hybridization step
1. organization chip is put in baking oven, temperature is adjusted to 60-65 degree, dries wax 1 hour；
2. the pretreatment of probe：
Prepare 1 × miRNA-ISH buffer solutions：2 × miRNA-ISH of 10ml buffer solutions and 10ml DEPC H2O, by the core of 40ul
Acid probe is placed in the PCR pipe without RNase, 90 DEG C of denaturation 4min, is placed on ice, add after quick centrifugation 20ml 1 ×
MiRNA-ISH is buffered, and is dispensed by the volume of 1-2ml, and direct freeze thawing when to be used is used；
3. buffer：
10×PBS：40g NaCl, 1g KCl, 7.2g Na2PO4,1.2g KH2PO4 are taken, with HCl pH 7.4 is adjusted, use DEPC
Water is settled to 500ml；
1×PBT：50ml 10 × PBS and 0.5ml Tween are taken, with DEPC water 500ml is settled to；
1M Tris-HCl, PH7.4：30.475g Tris alkali, plus 150mlDEPC water, PH is adjusted to 7.4 with concentrated hydrochloric acid, uses DEPC
Water is settled to 250ml；
Proteinase K stores the preparation of liquid：With 10mM Tris-HCl PH.7.5 diluted protein enzyme K to 20mg/ml；
Maleic acid buffer solution：0.1M maleic acids, 0.15MNaCl adjusts PH to 7.5 with NaOH；
NBT/BCIP dilutions：100ml 1M/L Tris-HCl, 20ml 5M/L NaCl, 880ml DEPC ddH2O, pH9.5；
4. sample dewaxing：
3 each 5min of dimethylbenzene,
Ethanol embathes 10 times about 100%,
Ethanol embathes 10 times about 100%
Ethanol 100%5min,
Ethanol embathes 10 times about 96%,
Ethanol 96%5min,
Ethanol embathes 10 times about 70%,
Ethanol 70%5min,
PBS 2-5min；
5. protease digestion sample：
37 DEG C of Proteinase K 15ug/mL processes 10-30min, PBS 2 times, simply shakes；
6. dehydration of alcohol：
Ethanol embathes 10 times about 70%
Ethanol 70%1min,
Ethanol embathes 10 times about 96%,
Ethanol 96%1min,
Ethanol embathes 10 times about 100%,
Ethanol 100%1min, is placed on clean paper and is air-dried 15min；
7. hybridize：
Add the hybridization solution of 50-100ul on every slide；22mm × 22mm cover glasses are covered on slide, glue mounting is used, in hybridization
Temperature 50 C 1h；
8. it is strict to develop a film：
Mounting glue is torn with tweezers are careful, slide are put into following solution gradient and developed a film：
50 DEG C of 5 × SSC 5min hybridization temperatures,
50 DEG C of 1 × SSC 5min hybridization temperatures,
50 DEG C of 0.2 × SSC 5min hybridization temperatures,
0.2 × SSC 5min room temperatures,
Room temperature, 1 time × 5min of PBS；
9. immune detection：
Slide is dried, confining liquid is added dropwise in room temperature wet box, close 15min；Blotted after confining liquid with paper, be placed on wet box 1:
800anti-DIG-AP Fab fragments, 4 DEG C overnight；Room temperature, 3 times × 3min of PBST；
10. color reaction and mounting is redyed：
Lucifuge reaction 30 DEG C of colour developings 2h (400ul/ slides) of NBT/BCIP, room temperature, 3 times × 5min of PBST；Develop a film in water, 2
Secondary 1min；Plus 200ul core fast red dye liquors, -1 minute 10 seconds；Slice, thin piece is rinsed 10 minutes in running water, dehydration of alcohol；With in
Property resin mounting；
(3) in situ hybridization testing result judges：
By double-blind study independent evaluations, its positive cell number and dye levels, positive cell number scoring are evaluated respectively：Have no positive
Cell is 0, positive cell<5% is 1, and 6%～25% is 2, and 25%～50% is 3, and 51%～75% is 4,>75% is 5；Dye
Color degree scores：Have no that dyeing is 0, it is 1 light gray coloured particles occur, it is 2 Dark grey occur, it is 3 black particle occur, overall score
As a result=staining positive cells number scoring × dye levels scoring.
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| CN114410775A (en) * | 2022-02-28 | 2022-04-29 | 陈益丹 | Marker for evaluating curative effect of allergic rhinitis acupuncture and application thereof |
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| CN103866040A (en) * | 2014-04-04 | 2014-06-18 | 广州伯信生物科技有限公司 | FISH probe composition and kit for detecting miRNA gene related to esophagus cancer and use method of kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109988842A (en) * | 2019-04-19 | 2019-07-09 | 上海吉玛制药技术有限公司 | A kind of reagent set and application of oligonucleotide marker probe in-situ detection PDL1 |
| CN109988842B (en) * | 2019-04-19 | 2022-12-06 | 上海吉玛制药技术有限公司 | Reagent group for in-situ detection of PDL1 by oligonucleotide labeled probe and application thereof |
| CN114410775A (en) * | 2022-02-28 | 2022-04-29 | 陈益丹 | Marker for evaluating curative effect of allergic rhinitis acupuncture and application thereof |
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