CN104630348A

CN104630348A - RT-PCR primers for detecting transcriptional level of NOX1 mRNA in serum as well as kit and method for evaluating concurrent intestinal cancer susceptibility of hyperglycemia population

Info

Publication number: CN104630348A
Application number: CN201510027147.4A
Authority: CN
Inventors: 崔戈; 鲍鹰; 冯文明; 郭慧慧; 薛涛; 王翔
Original assignee: First Peoples Hospital of Huzhou
Current assignee: First Peoples Hospital of Huzhou
Priority date: 2015-01-20
Filing date: 2015-01-20
Publication date: 2015-05-20

Abstract

The invention relates to an RT-PCR kit and a method for evaluating the concurrent intestinal cancer susceptibility of a hyperglycemia population by using the kit, belonging to the technical field of real-time fluorescence quantitative reverse transcription polymerase chain reaction kit development and human pathology combination. RT-PCR primers for detecting the transcriptional level of NOX1 mRNA in serum include a primer F: 5'-ATAGCAGAAGCCGACAGG-3' and a primer R: 5'-CCAGTGAGACCAGCAATG-3'. The invention establishes a method for detecting NOX1 mRNA in peripheral vein serum by using the specific primers; and a quantitative PCR amplification technology is adopted in the method, so that the sensitivity for detecting NOX1 mRNA is greatly improved, and enough information can be obtained in extremely few specimens.

Description

Detect the RT-PCR primer of serum NO X1 mRNA transcriptional level, the test kit assessing the concurrent intestinal cancer susceptibility of hyperglycemia population and appraisal procedure

Technical field

The present invention relates to a kind of method of primer, test kit and the concurrent colorectal cancer susceptibility of assessment hyperglycemia population; Particularly relate to a kind of detect serum NO X1 mRNA transcriptional level RT-PCR primer, for assessment of the RT-PCR kit of the concurrent colorectal cancer susceptibility of hyperglycemia population and the method for the concurrent colorectal cancer susceptibility of assessment hyperglycemia population.Belong to the technical field that Real time quantitative RT-PCR kit developing combines with Human pathology.

Background technology

According to the World Health Organization's statistics, the whole world exceedes 2.2 hundred million people and suffers from diabetes, within 2004, has 3,400,000 people to die from the severe complication of hyperglycemia initiation, and to the year two thousand thirty, because suffering from diabetes, the number of death will double again.In the Chinese population of Ling≤20 year old, diabetes and pre-diabetes morbidity are respectively 9.7% and 15.5%, in prediction on such basis at present in state-owned nearly 100,000,000 grownups suffer from diabetes, about 1.5 hundred million grownups are in pre-diabetes, and China has become the country that diabetic subject is maximum in the world.

Colorectal cancer (colorectal cancer, CRC) is one of modal malignant tumour in world wide, and annual about 1,000,000 new cases in the whole world, occupy 3rd ~ 5 at Cancer Mortality, and in ascendant trend year by year; Its mortality ratio is about 20.51%, occupies 4th ~ 5; The annual new cases of China's colorectal cancer are up to 130,000 ~ 160,000 people, current morbidity is up to 69.9/10 ten thousand, one of China's three large cancers are become, " 2012 Chinese tumours registration annual report " that whole nation tumour Register issues add up colorectal cancer reached tumor incidence, the 5th of mortality ratio, and to raise year by year.

Domestic and international multinomial epidemiology confirms that diabetes and colorectal cancer incidence rate and prognosis are closely related, and hyperglycemia state is considered to one of important risk factor of Colorectal Cancer and progress.Diabetes especially diabetes B and colorectal cancer have many common onset risk factors, as obesity, full diet, shortage motion etc.The high risk population easily suffering from colorectal cancer may be become for some hyperglycemia population (especially T2DM patient).Long-term hyperglycemia state by activating some genetic expression, and then can take part in developing of colorectal cancer.Therefore, filter out the diabetes complicated colorectal cancer molecular marker that can be used for the early warning of hyperglycemia population concurrent colorectal cancer susceptibility, the course of disease and prognosis evaluation, and then carry out early examining targetedly early controlling, the course of disease of tumour patient and Index for diagnosis, treatment plan are formulated, survival rate improve and the improvement of quality of life all significant.

Within 1983, M μ llis has invented polymerase chain reaction (polymerase chain reaction, PCR) technology.Round pcr is a kind of isothermal DNA amplification, have special, responsive, productive rate is high, the outstanding advantages such as quick, easy.Application round pcr can make specific gene or DNA fragmentation, and amplification in vitro is hundreds thousand of to 1,000,000 times in a short period of time.Due to this technology have easy fast, specificity and the outstanding advantages such as highly sensitive, reproducible, be therefore widely used in fundamental research and clinical diagnosis.But traditional PCR technology also exists in the application can not carry out accurate quantitative analysis to specific mRNA, and easily pollute in operating process and make the shortcomings such as false positive rate is high, therefore can not meet real work needs, make it apply and be restricted.

Along with the appearance of real-time fluorescence quantitative PCR (real time quantitative PCR, qPCR), people really can accomplish the accurate quantification to trace mrna in PCR sample.QPCR adds fluorophor in PCR reaction system, utilizes fluorescent signal to accumulate and realizes the whole PCR process of Real-Time Monitoring, and can carry out quantitative analysis to starting template.RT-PCR needs fluorescent probe to participate in, and adds a pair primer sequence containing testing gene particular bases in the reaction system of PCR, this primer sequence can with template nucleic acid generation specific hybrid.PCR often copies a nucleotide fragments, and just have a fluorescent signal to be released, product and fluorescent signal produce man-to-man corresponding relation.Along with the increase of product, fluorescent signal also strengthens thereupon, when signal is strengthened to a certain threshold value (according to mean value and the average standard deviation of fluorescent signal baseline, the degree of confidence calculating 99.7% is greater than the fluorescent value of mean value, be threshold value), cycle index now and cycle threshold Ct (cycle threshold, Ct) go on record.In this loop parameter Ct and PCR system starting template number logarithm between have strict linear relationship.The Ct value of positive quantitative criterion template amplification of different gradient and the template number of this positive quantitative criterion is utilized to make typical curve through logistic fit, just can determine the quantity of starting template again according to the Ct value of testing sample accurately, therefore can calculate the quantity of original template according to the fluorescence intensity of PCR product.Real-time fluorescence quantitative PCR have high specificity, highly sensitive, quantitatively accurately, the advantage such as operational safety, using the method specific amplification mRNA, is a kind of highly sensitive, high specific, method easy and simple to handle.

Therefore, this area urgently wishes to utilize RT-qPCR technology the state of hyperglycemia population to be monitored, to easily assess susceptibility and the prognosis of the concurrent colorectal cancer of hyperglycemia population when the time comes, contribute to the early warning of diabetes complicated colorectal cancer patients, early examine early control, prognosis evaluation, for reduction China diabetes, the high incidence of colorectal cancer, high lethality rate, there is great using value.

Summary of the invention

In order to solve the problems of the technologies described above, the invention provides the RT-PCR primer detecting serum NO X1 mRNA transcriptional level.

The technical scheme that the present invention solves the problem is as follows:

Detect the RT-PCR primer of serum NO X1 mRNA transcriptional level:

Primers F: 5 '-ATAGCAGAAGCCGACAGG-3 '; Primer R:5 '-CCAGTGAGACCAGCAATG-3 '.

Of the present inventionly additionally provide another kind of technical scheme, as follows:

Detect serum NO X1 mRNA transcriptional level for assessment of the RT-PCR kit of the concurrent colorectal cancer susceptibility of hyperglycemia population, comprise above-mentioned RT-PCR primer.

Preferred as technique scheme, also comprise internal reference primer, described internal reference primer is as follows:

Primers F: 5'-CACCCACTCCTCCACCTTTG-3';

Primer R:5'-CCACCACCCTGTTGCTGTAG-3'.

Preferred as technique scheme, also comprises RNA and extracts reagent, reverse transcription reagents and PCR reaction solution;

Component and the concentration of described RNA extraction reagent are as follows: Trizol reagent 355 ~ 800ml, chloroform 200 ~ 400 μ L, Virahol 600 ~ 1000 μ L, dehydrated alcohol 1.75 ~ 5.00mL, DEPC water 290 ~ 1000 μ L;

Described reverse transcription reagents component and concentration as follows: 150 ~ 250U/ μ L reversed transcriptive enzyme 2 μ L, 4 ~ 6 × RT Buffer 4 μ L, 12 ~ 50 mmol/L dNTPs 4 μ L, 0.1 ~ 1.2mol/L DTT 2 μ L, 35 ~ 45U/ μ L RNA enzyme inhibitors 0.5 μ L;

Described PCR reaction solution component and concentration as follows: Syber Green I Fluorescent nucleic acid stain 25 μ L, archaeal dna polymerase 100U/ml, dNTPs 0.2 mmol/L, magnesium chloride 6 mmol/L, Tri(Hydroxymethyl) Amino Methane Hydrochloride 16.5 mmol/L, Repone K 89.3 mmol/L, DEPC water 50 μ L ~ 60 μ L.

Preferred as technique scheme, described Trizol reagent is specially: heavily steam phenol 200mL, oxine 0.2g, β-mercaptoethanol 1.2g/dithiothreitol (DTT) 0.5g, different sulphur hydracid guanidine 100g, distilled water 118mL, 0.75M Trisodium Citrate 7.04mL, 10% sarcosyl 10.56mL and 2M sodium-acetate 20mL.

Preferred as technique scheme, reverse transcription reaction system is as follows:

Primers F 1 μ L,

Primer R 1 μ L,

RNA template 2 μ L,

Reversed transcriptive enzyme 2 μ L,

5 × RT Buffer 4 μ L,

25mM dNTPs 4μL、

0.1 M DTT 2μL、

RNA enzyme inhibitors 0.5 μ L

DEPC water surplus

Cumulative volume 20 μ L.

Preferred as technique scheme, amplification system is as follows:

Primers F 1 μ L,

Primer R 1 μ L,

CDNA template 2 μ L,

SYBR Green I Fluorescent nucleic acid stain 2 μ L

DEPC water surplus

Cumulative volume 50 μ L.

Another object of the present invention is to provide the method assessing the concurrent colorectal cancer susceptibility of hyperglycemia population by detecting serum NO X1 mRNA transcriptional level, and its technical scheme is as follows:

Assessing the method for the concurrent colorectal cancer susceptibility of hyperglycemia population by detecting serum NO X1 mRNA transcriptional level, comprising the following steps:

One, the extraction of serum total serum IgE

(1) collect the peripheric venous blood 5ml on an empty stomach in early morning of evaluation object, be placed in refrigerated centrifuge, lower turn over speed 2500-3000 r/min, after centrifugal 8-10min, draw serum 2 ml ~ 3ml with aseptic straw for 4 DEG C;

(2) above-mentioned serum is added in the homogenate tube containing 1mL Trizol reagent, in refiner homogenate 20sec, be put in immediately on ice;

(3) super clean bench is placed in, incubation 5min, 12000r/min, centrifugal 10min;

(4) suct clearly in new 1.5mL centrifuge tube, add the chloroform of 200 μ L, shake up, room temperature leaves standstill 2min, 4 DEG C, 12000r/min, centrifugal 10min;

(5) draw supernatant in new 1.5mL centrifuge tube, add the Virahol of 600 μ L, mix, room temperature leaves standstill 15min, 4 DEG C, 12000r/min, and centrifugal 15min, abandons supernatant;

(6) add the dehydrated alcohol rinsing precipitation of 1mL75%, 4 DEG C, 12000r/min, centrifugal 5min, abandons supernatant; The dehydrated alcohol of described 75% consist of 750 μ L dehydrated alcohol+250 μ L DEPC water;

(7) add 1mL dehydrated alcohol, rinsing precipitates, 4 DEG C, 12000r/min, and centrifugal 5min, abandons supernatant, drying at room temperature 10min;

(8) add the DEPC water dissolution RNA of 40 μ L, be placed in-80 DEG C of Refrigerator stores for subsequent use;

Two, reverse transcription synthesis cDNA

Above-mentioned RT-PCR kit is adopted to carry out reverse transcription, response procedures: 42 DEG C, 50min; 85 DEG C, 5min; Reaction terminates rear centrifugal, is placed in-20 DEG C of preservations;

Three, Real-time pcr amplification

Adopt above-mentioned RT-PCR kit to increase, response procedures is as follows: 95 DEG C, 10min; 95 DEG C, 15 Sec; 60 DEG C, 1min; 95 DEG C, 15 Sec; 60 DEG C, 15 Sec;

Four, destination gene expression is verified

The relative expression quantity of analytic statistics NOX1 mRNA, adopts SDS 2.3 software analysis result, the cycle number of Ct for experiencing when the fluorescent signal in each reaction tubes arrives the threshold value of setting, adopts and compares NOX1 mRNA relative expression quantity in Ct value method calculating sample; Calculate 2^ (-△ Ct) value and represent the initial mRNA relative expression quantity of goal gene in sample, △ Ct is that in same sample, goal gene Ct value deducts internal reference Ct value; Gene expression amount is converted into by 2^ (-△ Ct); Be corrected to 1 as reference using the 2^ of sample 2 (-△ Ct) value, calculate the relative expression quantity of goal gene; Use SPSS19.0 statistical software, enumeration data is with mean ± standard deviation represent; With p< 0.05 has statistical significance for difference, with p< 0.01 is remarkable statistical significance for difference has.

Aforesaid method of the present invention does not belong to the Clinics and Practices method of the disease of patent law the 3rd section of 25 articles regulation.Reason is as follows: one, and the method is implemented in vitro; They are two years old, according to the result that the method draws, quality all can not directly as judging disease whether index, this result can only for assessment of specific crowd (diabetic subject, especially diabetes B patient) suffer from the future the possibility of colorectal cancer, and the assessment to its present health condition, contribute to the early warning of diabetes complicated colorectal cancer, early examine early control, prognosis evaluation.

There is not yet the report of the specificity molecular marker about the concurrent colorectal cancer of hyperglycemia state at present both at home and abroad.

This study group of NOX1NOX1NOX1NOX1 have collected the first fasting blood sugar of being admitted to hospital of 391 colorectal cancer patients, adds up different glucose level colorectal cancer patients percentage, and analyses the dependency of different glucose level and clinicopathologic features.We find, the concurrent colorectal cancer patients of hyperglycemia is up to 29.67%, and the patient tumors diameter of the concurrent colorectal cancer of hyperglycemia is larger, differentiation degree is poorer, and prompting glucose level obviously can affect malignancy and the progress extent of colorectal cancer patients.

This study group adopts Illumina Hiseq s-generation order-checking platform to carry out the order-checking of high-throughput transcript profile respectively to colorectal cancer tumor tissues that is concurrent and not complicated with hyperglycemia and normal bowel tissue samples, after bioinformatics analysis, preliminary screening is a collection of at the diabetes complicated Expression in Colorectal Cancer measurer molecular marker that there were significant differences.For verifying the reliability of the molecular marker of above-mentioned screening for the concurrent colorectal cancer course of disease of hyperglycemia and prognosis evaluation further, we have collected 120 routine colorectal cancer patients tumours, healthy tissues and serum sample, have collected 120 routine Diabetes Mellitus samples, to detect when being admitted to hospital at the beginning of above-mentioned two class patients fasting blood glucose level in early morning, after glucose level and good pernicious classification, real-time quantitative PCR method is adopted to demonstrate the mRNA level in-site of specificity molecular marker in above-mentioned two class patient correlated sampleses further.

Our the result finds, nadph oxidase 1(Homo sapiens NADPH oxidase 1, NOX1) expression level in the colorectal cancer patients tumor tissues of complicated with hyperglycemia is significantly higher than the normal bowel tissue of complicated with hyperglycemia colorectal cancer patients, and the expression level in the colorectal cancer patients tumor tissues of euglycemia is significantly higher than the colorectal cancer patients normal bowel tissue of euglycemia.And do not express or low expression in normal colonic mucosa cell and healthy population peripheral blood.We think that NOX1 has higher specificity and susceptibility in the developing of concurrent colorectal cancer under hyperglycemia state, can as one of mark of diabetes complicated colorectal cancer.

Recent study shows, reactive oxygen species (reactive oxygen species, ROS) be the signal of interest molecule that the differentiation of participation cells in vivo, propagation, conversion, apoptosis and vasculogenesis regulate, in cell, ROS is mainly generated by nadph oxidase regulation and control.NOX1 increases the ability of tumour cell opposing apoptosis by producing ROS thus promotes growth of tumour cell.The height of NOX1 activity is relevant with colon cancer cell Infiltration and metastasis ability to have scholar to report recently, and NOX1 may play an important role in the generation, development of colorectal cancer.Separately there are some researches show, it is the impaired key link of diabetes inner skin cell function that eNOS solution is coupled, and eNOS solution is coupled and activates relevant with NOX1 subunit.

In sum, the present invention has following beneficial effect:

1, product of the present invention will by detecting NOX1 mRNA expression level in hyperglycemia state crowd peripheral vein serum sample basis, can be used for susceptibility and the prognosis of assessing the concurrent colorectal cancer of hyperglycemia population, contribute to the early warning of diabetes complicated colorectal cancer patients, early examine early control, prognosis evaluation, for reduction China diabetes, the high incidence of colorectal cancer, high lethality rate, there is great using value;

2, the present invention establishes the method utilizing Auele Specific Primer to detect peripheral vein serum NOX1 mRNA; Because present method have employed quantitative PCR amplification technique, the susceptibility detecting NOX1 mRNA is improved greatly, can ensure to obtain enough information in few sample;

3, present invention also offers the cDNA template (volume 2 μ L) containing high expression level NOX1 gene as positive control (standard substance); Standard substance base sequence is: CTATATCCTTGGCTTAGGGATTCACGGCATTGGTGGAATTGTCCGGGGTCAAACAG AGGAGAGCATGAATGAGAGTCATCCTCGCAAGTGTGCAGAGTCTTTTGAGATGTGG GATGATCGTGACTCCCACTGTAGGCGCCCTAAGTTTGAAGGGCATCCCCCTGAGTC TTGGAAGTGG;

4, present invention also offers not expressing or the low cDNA template (volume 2 μ L) expressing NOX1 gene as negative control.

Accompanying drawing explanation

What Fig. 1 showed is that serum sample of the present invention extracts total serum IgE agarose gel electrophoresis;

Fig. 2 display be the RNA sample integrity of Agilent 2100 chip detection sample 1 of the present invention;

Fig. 3 display be the RNA sample integrity of Agilent 2100 chip detection sample 2 of the present invention;

What Fig. 4 showed is NOX1 mRNA of the present invention amplification kinetic curve;

What Fig. 5 showed is GAPDH mRNA of the present invention amplification kinetic curve;

Fig. 6 display be the relative expression quantity of NOX1 mRNA of the present invention.

Embodiment

Below in conjunction with embodiment, the present invention is further described.But should be clear and definite, hereinbefore explanation and following embodiment are only as illustrating, do not form limitation of the scope of the invention.

The experimental technique used in following embodiment if no special instructions, is ordinary method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

1. design of primers and synthesis

1.1 NOX1 gene primer Design and synthesis

The NOX1 gene standard substance sequence intending amplification is NOX1 gene the 2nd, 3 exon 1 266-422 base, with NOX1 full length mRNA sequence for template design primer.Standard substance PCR upstream primer F sequence is: 5 '-ATAGCAGAAGCCGACAGG-3 '; Downstream primer R sequence is: 5 '-CCAGTGAGACCAGCAATG-3 '.

1.2 GAPDH gene primer Design and synthesis

Internal reference gene mankind glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequence intending amplification is GAPDH gene the 6th, 7 exon 1 1065-1174 base, with the long mRNA sequence of GAPDH for template design primer, GAPDH upstream region of gene primers F sequence is: 5'-CACCCAC TCCTCCACCTTTG-3'; Downstream primer R sequence is: 5' – CCACCACCCTGTTGCTGTAG-3'.

2. contain the cDNA template of high expression level NOX1 gene, do not express or low cDNA method for preparing template of expressing NOX1 gene:

The preparation of 2.1 associated reagents:

(1) DEPC water compound method:

At the dual distilled water (ddH of 1000mL ₂o) add 1mLDEPC stoste in, magnetic stirrer is spent the night.

(2) 1 mol/L TrisCl(pH=8.0) compound method: dissolve 121.1 g Tris in 800 mL distilled water, enriching HCl adjusts pH to 8.0, and be settled to 1 L, autoclaving is for subsequent use.

(3) 50 × TAE buffer methods: dissolve 242 g Tris in 500 mL distilled waters, add 100 mL 0.5 mol/L EDTA (pH=8.0), 57.1 mL glacial acetic acids, be settled to 1 L, room temperature preservation is for subsequent use.

The extraction of 2.2 serum total serum IgE

(1) with EDTA-K ²the each 5ml(of empty stomach peripheric venous blood in early morning of anti-freezing vacuum negative pressure blood-taking pipe collection specific crowd 1, specific crowd 2 is respectively sample 1, sample 2), be placed in refrigerated centrifuge, lower turn over speed 2500-3000 r/min, after centrifugal 8-10min, draw serum 2 ml ~ 3ml with aseptic straw for 4 DEG C;

(note: specific crowd 1: the concurrent Colon and rectum patient of hyperglycemia: such patient annual fasting blood glucose level >7.77mmol/L, tumor tissues when not using antidiabetic drug are diagnosed as colorectal cancer through Pathology Deparment after excision; Specific crowd 2: diabetes (non-accompanying tumors sick) patient: this patient when not using antidiabetic drug monthly average fasting blood glucose level >7.77mmol/L, check without colorectal carcinoma person through Ultrasonic tomography/intestines mirror biopsy pathology)

(2) above-mentioned serum is added in the homogenate tube containing 1mL Trizol, in refiner homogenate 20sec, be put in immediately on ice;

(6) add dehydrated alcohol (750 μ L dehydrated alcohol+250 μ L DEPC water) the rinsing precipitation of 1mL75%, 4 DEG C, 12000r/min, centrifugal 5min, abandons supernatant;

(8) add the DEPC water dissolution RNA of 40 μ L, be placed in-80 DEG C of Refrigerator stores for subsequent use.

2.3 RNA quality examinations

(1) agarose gel electrophoresis: 1% agarose gel electrophoresis, 150V voltage 15 minutes.Observe under ultraviolet transmission light and take pictures.

Agarose gel electrophoresis the results are shown in Figure 1:

(note: the RNA electrophoresis that sample 1, sample 2 extract is respectively No. 11, No. 12 bands)

Result illustrates: 28S and 18S ribosomic RNA bands very bright and dense (its size is decided by the species for extracting RNA).Well be positioned at above picture see chart-pattern under, above the density of a band (eucaryon 28S, protokaryon 23S) be approximately 2 times an of band (eucaryon 18S, protokaryon 16S) below.Less band spread a little is also likely observed in below, and it is by low-molecular-weight RNA(tRNA and 5S ribosome-RNA(rRNA) etc.) form.Between 28S and 18S rrna band, generally can see the EB coloring matter of a slice disperse, may be made up of mRNA and other special-shaped RNA.

(2) NanoDrop 2000 detects RNA concentration and OD value:

Get 1 μ 1RNA sample and be placed in nanodrop microanalyser pedestal, detection by quantitative wavelength, in the OD value of 260,280, verifies RNA purity.OD(260/280) reading is purer sample between 1.80-2.00.RNA concentration and total amount are required: RNA concentration >=200 ng/ μ l; RNA demand >=10 μ g.

Result is as follows:

Sample 1:OD(260/280) reading 1.95, RNA concentration 407ng/ μ l;

Sample 2:OD(260/280) reading 1.90, RNA concentration 389ng/ μ l;

(3) Agilent 2100 chip detection:

Operate according to RNA 6000 Nano chip agent box.Respectively sampling this 1,9 μ l total serum IgE samples of sample 2 add the hole for injecting glue of electrophoresis chip after mixing with dyestuff.In Ladder hole, add 1 μ l Ladder, in the sample well of numerical markings, respectively add 1 μ l sample.The RNA sample integrity detection of sample 1, sample 2 the results are shown in Figure 2, Fig. 3:

Result illustrates: the concentration degree at each peak in figure, RNA integrity degree in representative sample, peak more concentrated representative sample RNA degradation rate is lower.The reading of rRNA Ratio [28s/18s], represents the concentration ratio of 28S and 18S.RIN value represents the integrity degree of RNA sample, and numerical value is at 1-10, and numerical value is higher, and to represent RNA integrity better.Generally, RIN value >6.5 is qualified, and RIN value≤6.5 are defective.The proportionlity etc. of species, sample viscosity and 28S and 18S, 5S all can affect detection data results, and instrument even can be caused to report an error.

2.4 reverse transcription synthesis cDNA

Use RT-PCR test kit, carry out reverse transcription with 20 μ l total reaction volume.Add by following system:

Response procedures: 42 DEG C, 50min; 85 DEG C, 5min; Reaction terminates rear of short duration centrifugal, is placed in-20 DEG C of preservations.

2.5 Real-time pcr amplification checking destination gene expressions

The cDNA prepared is carried out pcr amplification, and amplification system is as follows:

Response procedures: 95 DEG C, 10min(95 DEG C, 15Sec; 60 DEG C, 1min) × 40; 95 DEG C, 15 Sec; 60 DEG C, 1min; 95 DEG C, 15 Sec; 60 DEG C, 15 Sec.

Data acquisition instrument carries software analysis: ABI Prism 7300 SDS Software.

NOX1 mRNA amplification kinetic curve is shown in Fig. 4, and GAPDH mRNA amplification kinetic curve is shown in Fig. 5.

2.6 result statistical study

The relative expression quantity of analytic statistics NOX1 mRNA, adopts SDS 2.3 software analysis result.The cycle number (cycle threshold) of Ct for experiencing when the fluorescent signal in each reaction tubes arrives the threshold value of setting.Employing is compared Ct value method and is calculated NOX1 mRNA relative expression quantity in sample.Calculate 2^ (-△ Ct) value and represent the initial mRNA relative expression quantity of goal gene in sample.△ Ct is that in same sample, goal gene Ct value deducts internal reference Ct value; Gene expression amount is converted into by 2^ (-△ Ct); Be corrected to 1 as reference using the 2^ of sample 2 (-△ Ct) value, calculate the relative expression quantity of goal gene.Use SPSS19.0 statistical software, enumeration data is with mean ± standard deviation represent.With p< 0.05 has statistical significance for difference, with p< 0.01 is remarkable statistical significance for difference has.

Statistics see Fig. 6 (sample 1 compares with sample 2, p< 0.01)

Note: sample 1 is diabetes complicated serum in patients with colorectal sample;

Sample 2 is diabetes (non-accompanying tumors is sick) patients serum's sample.

Sample 1 is the NOX1 mRNA relative expression quantity of diabetes complicated serum in patients with colorectal sample, and sample 2 is the NOX1 mRNA relative expression quantity of diabetes (not concurrent colorectal cancer) patients serum's sample.Result shows: the NOX1 mRNA relative expression quantity of diabetes complicated serum in patients with colorectal sample is significantly higher than the NOX1 mRNA relative expression quantity of diabetes (non-accompanying tumors is sick) patients serum's sample.

SEQUENCE LISTING

<110> The First People's Hospital of Huzhou

<120> detect serum NO X1 mRNA transcriptional level RT-PCR primer, for assessment of the RT-PCR kit of the concurrent colorectal cancer susceptibility of hyperglycemia population and the method for the concurrent colorectal cancer susceptibility of assessment hyperglycemia population

<130>

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 18

<212> DNA

<213> artificial sequence

<400> 1

atagcagaag ccgacagg 18

<210> 2

<211> 18

<212> DNA

<213> artificial sequence

<400> 2

ccagtgagac cagcaatg 18

<210> 3

<211> 20

<212> DNA

<213> artificial sequence

<400> 3

cacccactcc tccacctttg 20

<210> 4

<211> 20

<212> DNA

<213> artificial sequence

<400> 4

ccaccaccct gttgctgtag 20

Claims

1. detect the RT-PCR primer of serum NO X1 mRNA transcriptional level, it is characterized in that:

Primers F: 5 '-ATAGCAGAAGCCGACAGG-3 ';

Primer R:5 '-CCAGTGAGACCAGCAATG-3 '.

2. detect serum NO X1 mRNA transcriptional level for assessment of the RT-PCR kit of the concurrent colorectal cancer susceptibility of hyperglycemia population, it is characterized in that comprising RT-PCR primer according to claim 1.

3. RT-PCR kit according to claim 2, is characterized in that also comprising internal reference primer, and described internal reference primer is as follows:

Primers F: 5'-CACCCACTCCTCCACCTTTG-3';

Primer R:5'-CCACCACCCTGTTGCTGTAG-3'.

4. RT-PCR kit according to claim 2, is characterized in that also comprising RNA extracts reagent, reverse transcription reagents and PCR reaction solution;

5. RT-PCR kit according to claim 4, is characterized in that described Trizol reagent is specially: heavily steam phenol 200mL, oxine 0.2g, β-mercaptoethanol 1.2g/dithiothreitol (DTT) 0.5g, different sulphur hydracid guanidine 100g, distilled water 118mL, 0.75M Trisodium Citrate 7.04mL, 10% sarcosyl 10.56mL and 2M sodium-acetate 20mL.

6. RT-PCR kit according to claim 2, is characterized in that reverse transcription reaction system is as follows:

Primers F 1 μ L,

Primer R 1 μ L,

RNA template 2 μ L,

Reversed transcriptive enzyme 2 μ L,

5 × RT Buffer 4 μ L,

25mM dNTPs 4μL、

0.1 M DTT 2μL、

RNA enzyme inhibitors 0.5 μ L

DEPC water surplus

Cumulative volume 20 μ L.

7. RT-PCR kit according to claim 2, is characterized in that amplification system is as follows:

Primers F 1 μ L,

Primer R 1 μ L,

CDNA template 2 μ L,

SYBR Green I Fluorescent nucleic acid stain 2 μ L

DEPC water surplus

Cumulative volume 50 μ L.

8. assessing the method for the concurrent colorectal cancer susceptibility of hyperglycemia population by detecting serum NO X1 mRNA transcriptional level, comprising the following steps:

The extraction of serum total serum IgE

Collect the peripheric venous blood 5ml on an empty stomach in early morning of evaluation object, be placed in refrigerated centrifuge, lower turn over speed 2500-3000 r/min, after centrifugal 8-10min, draw serum 2 ml ~ 3ml with aseptic straw for 4 DEG C;

Above-mentioned serum is added in the homogenate tube containing 1mL Trizol reagent, in refiner homogenate 20sec, be put in immediately on ice;

Be placed in super clean bench, incubation 5min, 12000r/min, centrifugal 10min;

Suct clearly in new 1.5mL centrifuge tube, add the chloroform of 200 μ L, shake up, room temperature leaves standstill 2min, 4 DEG C, 12000r/min, centrifugal 10min;

Draw supernatant in new 1.5mL centrifuge tube, add the Virahol of 600 μ L, mix, room temperature leaves standstill 15min, 4 DEG C, 12000r/min, and centrifugal 15min, abandons supernatant;

Add the dehydrated alcohol rinsing precipitation of 1mL75%, 4 DEG C, 12000r/min, centrifugal 5min, abandons supernatant; The dehydrated alcohol of described 75% consist of 750 μ L dehydrated alcohol+250 μ L DEPC water;

Add 1mL dehydrated alcohol, rinsing precipitates, 4 DEG C, 12000r/min, and centrifugal 5min, abandons supernatant, drying at room temperature 10min;

Add the DEPC water dissolution RNA of 40 μ L, be placed in-80 DEG C of Refrigerator stores for subsequent use;

Reverse transcription synthesis cDNA

RT-PCR kit according to claim 6 is adopted to carry out reverse transcription, response procedures: 42 DEG C, 50min; 85 DEG C, 5min; Reaction terminates rear centrifugal, is placed in-20 DEG C of preservations;

Real-time pcr amplification

Adopt RT-PCR kit described in claim 7 to increase, response procedures is as follows: 95 DEG C, 10min; 95 DEG C, 15 Sec; 60 DEG C, 1min; 95 DEG C, 15 Sec; 60 DEG C, 15 Sec;

Checking destination gene expression