CN107604068A

CN107604068A - A kind of kit that carcinoma of urinary bladder is detected using long-chain non-coding RNA

Info

Publication number: CN107604068A
Application number: CN201711002908.6A
Authority: CN
Inventors: 李翀; 李书成
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-10-24
Filing date: 2017-10-24
Publication date: 2018-01-19
Anticipated expiration: 2037-10-24
Also published as: CN107604068B

Abstract

The invention discloses a kind of kit that carcinoma of urinary bladder is detected using long-chain non-coding RNA, the kit includes：The anti-sense primer as described in sense primer and SEQ ID No.3 as described in SEQ ID No.2；Probe；Include the standard DNA template of nucleotide sequence described in SEQ ID No.1；PCR reaction solutions.The kit that carcinoma of urinary bladder is detected using long-chain non-coding RNA of the present invention, detections of the long-chain non-coding RNA lncRNA TMP125 to Bladder Cancer is selected, it is good to the Detection results of carcinoma of urinary bladder, there are higher sensitivity and specificity.

Description

A kind of kit that carcinoma of urinary bladder is detected using long-chain non-coding RNA

Technical field

The present invention relates to detection kit technical field, and in particular to a kind of carcinoma of urinary bladder gene detecting kit.

Background technology

Carcinoma of urinary bladder is the most common malignant tumour of urinary system, and in China, the incidence of disease occupies first of Patients with Urinary System Tumors.Wing Guang cancer easily recurs, and excavates a kind of novel bladder carcinoma marker that can be used for early screening and Prognosis scoveillance, it appears particularly important. Long-chain non-coding RNA (lncRNA) refers to more than 200 nucleotides of length, has the non-coding RNA of controlling gene expressional function. Research finds that lncRNA take part in intracellular a variety of important vital movements, though without protein coding function, can pass through apparent something lost Modification controlling gene expression is passed, the generation, development with tumour are inseparable.In recent years, the relation of lncRNA and carcinoma of urinary bladder also by Gradually attract attention, some there is particularly important lncRNA to be expected to as the biomarker of Diagnosis of Bladder and the molecule for the treatment of Target spot.

In the detection of traditional carcinoma of urinary bladder, marks of the lncRNA as detection is seldom used, although by largely grinding Study carefully, but still for find effectively using lncRNA as detection mark, it would be highly desirable to further research and development one kind using lncRNA as Detect the detection kit of mark.

The content of the invention

It is an object of the invention to provide a kind of kit that carcinoma of urinary bladder is detected using long-chain non-coding RNA, to solve The defects of present in prior art to specificity and the poor sensitivity of carcinoma of urinary bladder detection.

In order to solve the above technical problems, the present invention provides a kind of kit that carcinoma of urinary bladder is detected using long-chain non-coding RNA,

The kit includes：

The anti-sense primer as described in sense primer and SEQ ID No.3 as described in SEQ ID No.2；

Fluorescent marker；

Include the standard DNA template of nucleotide sequence described in SEQ ID No.1；

PCR reaction solutions.

Preferably, the PCR reaction solutions include 2 × Super TaqMan Onestep Buffer, Super Onestep Enzyme, probe, RNase-Free Water.

Preferably, PCR reaction systems are 25ul reaction systems, wherein, 2xSuper TaqMan Onestep Buffer 12.5ul；Sense primer, 10uM, 0.5ul；Anti-sense primer, 10uM, 0.5ul；Probe, 10pM, 0.5ul；Super Onestep Enzyme, 1.0ul；RNA Template, 10pg-100ng.

Preferably, the standard form DNA is transformed into DH5 α competent cells, contains SEQ ID No.1 for extraction The plasmid of shown sequence.

Preferably, the fluorescent marker be SYBR Green, TaqMan probe, molecular beacon, double cross probe or multiple Close probe.

Preferably, the TaqMan probe sequence is as shown in SEQ ID No.4.

Preferably, the kit also includes RNA extracts reagent, and the extracts reagent of the RNA is Thermo Fisher The TRIzol reagents of Scientific companies.

Preferably, the kit also includes nucleic acid quantification instrument.

Preferably, the kit also includes fluorescent quantitation amplification instrument.

The invention has the advantages that：

The present invention utilizes long-chain non-coding chip of expression spectrum technology, by variance analysis, screens in human bladder carcinoma tissue In the long-chain non-coding RNA significantly expressed, be named as lncRNA.The expression quantity of the long-chain non-coding RNA in cancerous tissue with just Often the expression quantity in tissue is compared, lncRNA-TMP125 significantly high expression in human bladder carcinoma tissue, and of the invention utilizes long-chain Non-coding RNA detects the kit of carcinoma of urinary bladder, selects inspections of the long-chain non-coding RNA lncRNA-TMP125 to Bladder Cancer Survey, it is good to the Detection results of carcinoma of urinary bladder, there are higher sensitivity and specificity.

Brief description of the drawings

Fig. 1 is the LncRNA chip testing results of the present invention, shows the top ten of high expression in Bladder Cancer LncRNA information.

Fig. 2 is to carry out Ago-Gel after the primer PCR of the sequences Design for lncRNA-TMP125 of the present invention expands Electrophoresis result figure.

Fig. 3 is that the lncRNA-TMP125 of 53 carcinoma of urinary bladder clinical samples of the present invention verifies qRT-PCR testing results.

Embodiment

Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.

The human bladder cancer of embodiment 1 and the lncRNA chip expression analysis of cancer beside organism, screen significantly high expression in cancerous tissue LncRNA

First, material and method

1. material

Tissue samples come from the surgery excision sample of 3 pairs of bladder cancer patients, and each pair includes Bladder Cancer and pairing Cancer beside organism.

2. method

(1) by TRIzol reagents (article No. 15596026) the extraction carcinoma of urinary bladder of Thermo Fisher Scientific companies Tissue and the total serum IgE of cancer beside organism.

(2) to sample RNA progress Cy5 fluorescence labelings, (Haikang is carried out into bioengineering Co., Ltd in commission " service is marked in ArrayStar Human LncRNA Microarray V4.0Service ")

(3) reverse transcription synthesizes the first chain cDNA：Using Total RNA as starting, the Oligo (dT) containing T7 promoter sequences Primer (going up Haikang into bioengineering Co., Ltd) is primer, uses the first chain of CbcScript enzymatic synthesis cDNA.

(4) the second chain cDNA is synthesized：Archaeal dna polymerase (upper Haikang into bioengineering Co., Ltd) using RNA fragments as primer, The second chain cDNA is synthesized, and purifies double-strand cDNA.

(5) in-vitro transcription synthesis cRNA：Using cDNA as template, using T7Enzyme Mix, (upper Haikang has into bioengineering Limit company) synthesis cRNA.

(6) random primed reverse transcription：1ug cRNA are taken, reverse transcription is carried out with random primer.

(7) hybridization and cleaning：CDNA is dissolved in 45 in hybridization solution (25% formamide, 5 × SSC, 0.1%SDS, 0.5%BSA) DEG C hybridized overnight, washed 5 minutes with SSC (upper Haikang into bioengineering Co., Ltd) buffer solution, slide can be used to after drying Scanning.

(8) chip scanning, graphical analysis, differential gene screening, chip Agilent Microarray Scanner (Agilent p/n G2565BA) is scanned, and is converted into data signal, and initial data is input into GeneSpring GX In software, differential gene screening is carried out.

2nd, result

As shown in figure 1, the lncRNA chip cluster analyses in the present invention on human bladder cancer, chip examination finds a plurality of table Up to the upper lncRNAs for reconciling and expressing and lowering.Wherein lncRNA-TMP125 is shown expresses significantly up-regulation in cancerous tissue.

Differences of the embodiment 2qRT-PCR preliminary identifications lncRNA-TMP125 in the cancerous tissue and cancer beside organism of carcinoma of urinary bladder Expression

First, experiment material

Cancerous tissue and the pairing cancer beside organism of 53 couples of human bladder cancers is chosen, lncRNA-TMP125 differential expression is carried out QRT-PCR is verified.

2nd, experimental method and result

1. primer specificity is identified

(1) from transcript sequence related Ensemble databases extraction lncRNA-TMP125, and according to the sequence of transcript Row design primer by NCBI design of primers instrument (Primer BLAST), upstream primer sequence as shown in SEQ ID No.2, And anti-sense primer is as shown in SEQ ID No.3.

(2) by human bladder carcinoma tissue and the TRIzol reagents of Thermo Fisher Scientific companies of cancer beside organism After (article No. 15596026) extraction total serum IgE, then with 7300real time PCR system nucleic acid quantification instrument (Applied Biosystems AB) quantitative extracted RNA purity and concentration.

(3) tried using Beijing CoWin Bioscience Co., Ltd. Super TaqMan OneStep RT-qPCR Kit Agent box (article No. CW2695), fluorescent quantitative PCR is carried out by template of RNA.

Quantitative fluorescent PCR system：

Reagent	25ul reaction systems	Final concentration
			2xSuper TaqMan Onestep Buffer	12.5ul	1x
Forward Primer, 10uM	0.5ul	0.2uM
			Reverse Primer, 10uM	0.5ul	0.2uM
Probe, 10pM	0.5ul	0.2uM
			Super Onestep Enzyme	1.0ul
RNA Template	X ul	10pg-100ng
			RNase-Free Water	Up to 25ul

Quantitative fluorescent PCR program is as follows：45 DEG C of reverse transcription, 20min；95 DEG C of PCR pre-degenerations, 2-5min；95 DEG C of denaturation, 15s, 60 DEG C, 30-45s of annealing/extension, common 30-40 circulation.

(4) electrophoresis detection, from DM2000DNA Marker (Beijing CoWin Bioscience Co., Ltd., article No. CW0632).As shown in Fig. 2 electrophoresis result is that amplified fragments size is identical with expection, amplified production only has a band.

2. liquid nitrogen grinding method is used, according to the TRIzol reagent (article No.s of Thermo Fisher Scientific companies 15596026) reagent needed for and the total serum IgE of step extraction Bladder Cancer or tumour.Main operational steps are as follows：

(1) fresh tissue sample is put into rapidly in the mortar equipped with liquid nitrogen and is ground, and is finally ground into powder；

(2) 1ml TRIzol reagents are added in each mortar, continues grinding 5 minutes, until forming homogenate shape；

(3) above-mentioned homogenate is transferred in the sterile no enzyme centrifuge tubes of 1.5ml, adds 0.2ml chloroforms in every 1 part of homogenate, mix 12000g is centrifuged 20 minutes after even, and RNA is present in upper strata aqueous phase；

(4) draw upper strata aqueous phase (about 200-300ul) to be transferred in the sterile no new pipes of enzyme of 1.5ml, 0.5ml isopropanols mix Even, 12000g is centrifuged 10 minutes；

(5) supernatant is abandoned, RNA precipitate is washed once with 75% ethanol, is dissolved several times with water piping and druming of the 20ul without RNase RNA, it is stored in -80 DEG C of low temperature refrigerators.

3. the preparation of standard DNA template

According to lncRNA-TMP125 base sequences shown in SEQ ID No.1, commission Shanghai life work synthetic DNA.

2ul synthetic products are sampled, being connected to pUC-TTA Cloning Kit, (Beijing health is the century limited public affairs of biotechnology Department, article No. CW2591), then it is transformed into DH5 α competent cells.By the primer as shown in SEQ ID No.2 and such as SEQ Primer screening positive colony shown in ID No.3, extract DNA, DNA 7300real time PCR system cores Sour quantitative instrument quantifies, and does 10 times and be serially diluted as standard curve that (standard DNA template concentration range is 10²-10⁶Copy/ u1)。

4. sensitivity experiments

Standard DNA template plasmid is diluted to 102 in proportion, 103,104,105,106 copies/u1, carry out fluorescent quantitation PCR, the detection sensitivity using the least concentration of test positive as this method.The method that this research is established, its detection range are 102-106 copies/u1.Concentration limit is 102 copies/u1.

5. fluorescence quantitative PCR detection lncRNA-TMP125 expression quantity

Fluorescent quantitative PCR, quantitative fluorescent PCR system are carried out by template of RNA：

According to qRT-PCR relative quantification formula：2^-△Ct, lncRNA-TMP125 is calculated respectively in bladder cancer patients cancer Organize the expression in (T) and cancer beside organism (N).As shown in figure 3, expression water of the lncRNA-TMP125 in cancer beside organism It is flat to be concentrated mainly between 0.37-3.28, and the expression quantity of the lncRNA-TMP125 in cancerous tissue is concentrated mainly on 1.053- Between 15.375, hence it is evident that higher than normal structure.Further confirm that lncRNA-TMP125 universal high expression in tumor tissues.This The as shown by data of embodiment：LncRNA-TMP125 is in 53 carcinomas of urinary bladder and cancer beside organism, 47 up-regulations, and positive rate= Up-regulated expression number of cases/always detect number of cases × 100%=47/53=88.7%, lncRNA-TMP125 can be used as Diagnosis of Bladder New tumor markers.

According to above-described embodiment 1 and the testing result of embodiment 2, using lncRNA-TMP125 long-chain non-coding RNAs in wing The characteristic of high expression in Guang cancerous tissue, its nucleotide sequence is as shown in SEQ ID No.1, and its transcript regions is located at No. 6 chromosomes, entirely Long 1122bp.Compared with normal structure, lncRNA-TMP125 significantly high expression in human bladder carcinoma tissue.The present invention provides one Using the kit of long-chain non-coding RNA detection carcinoma of urinary bladder, it is included such as SEQ ID No.1 sense primer and such as SEQ ID kind No.2 anti-sense primer；TaqMan probe sequence such as SEQ ID No.4；Include the standard DNA mould of SEQ ID No.1 nucleotide sequences Plate；PCR reaction solutions.

Wherein, PCR reaction solutions include 2xSuper TaqMan Onestep Buffer, Super Onestep Enzyme, RNase-Free Water, dNTP, Mg2+, Taq enzyme and buffer buffer solutions, sense primer and anti-sense primer.Fluorescence labeling Thing is SYBR Green II, TaqMan probe, molecular beacon, double cross probe or combined probe, and Taq enzyme is thermal starting enzyme.Inspection Test agent box is fluorescent quantificationally PCR detecting kit, and primer is applied to SYBR Green, TaqMan probe, molecular beacon, double miscellaneous Hand over the detection of probe, combined probe.

The present invention is as follows using the kit detecting step of long-chain non-coding RNA detection carcinoma of urinary bladder：

Step A：Extracted according to reagent needed for the TRIzol reagents of Thermo Fisher Scientific companies and step Total RNAs extraction sample total serum IgE, then with 7300real time PCR system nucleic acid quantifications instrument (Applied Biosystems AB) quantitative extracted purity and concentration.

Step B：Fluorescent quantitative PCR lncRNA-TMP125 is carried out by template of the total serum IgE of reverse transcription.

Specifically, the use of the detection carcinoma of urinary bladder lncRNA-TMP125 of present invention dye class PCR kit for fluorescence quantitative Method, quantitative fluorescent PCR reaction system are as follows：

Quantitative fluorescent PCR program is as follows：45 DEG C of reverse transcription, 20min；95 DEG C of PCR pre-degenerations, 2-5min；95 DEG C of denaturation, 15s, 60 DEG C, 30-45s of annealing/extension, common 30-40 circulation.By the detection to positive, quantification kit of the present invention Detection accuracy is 85.7%-91.6%, and continuous 3 repetitions are tested, and experimental result is stable.

The fluorescent quantificationally PCR detecting kit of the present invention is suitable for presently, there are all types fluorescent quantitation base of in the market Gene-amplification instrument, high sensitivity, specificity is good, has a good application prospect.

Embodiment 3 is using the detection kit of the present invention to lncRNA-TMP125 in Bladder Cancer and cancer beside organism Differential expression detected

First, experiment material

100 parts of human bladder carcinoma tissues and 100 parts of cancer beside organisms's (being provided by The Third Affiliated Hospital of Peking University) are provided, it is right LncRNA-TMP125 differential expression carries out qRT-PCR detections.

2nd, experimental method and result

1. the extraction of sample total serum IgE：

Using liquid nitrogen grinding method, with the TRIzol reagent (article No.s of Thermo Fisher Scientific companies 15596026) total serum IgE of Bladder Cancer or tumour is extracted.Main operational steps are as follows：

(2) 1ml TRIZOL reagents are added in each mortar, continues to grind 3-5 minutes, until into homogenate shape；

(3) above-mentioned homogenate is transferred in the sterile no enzyme centrifuge tubes of 1.5ml, adds 0.2ml chloroforms in every 1 part of homogenate, mix 12000g is centrifuged 10 minutes after even, and RNA is in upper strata aqueous phase；

2. fluorescent quantitative PCR

(1) sense primer as shown in SEQ ID No.2 and the anti-sense primer as shown in SEQ ID No.3 are used.

(2) human bladder carcinoma tissue and cancer beside organism are tried according to the TRIzol of Thermo Fisher Scientific companies After agent (article No. 15596026) extraction total serum IgE, then with 7300real time PCR system nucleic acid quantification instrument (Applied Biosystems AB) quantitative extracted RNA purity and concentration.

(3) fluorescent quantitative PCR is carried out by template of total serum IgE.

Quantitative fluorescent PCR reaction system：

The program of quantitative fluorescent PCR is as follows：45 DEG C of reverse transcription, 20min；95 DEG C of PCR pre-degenerations, 2-5min；95 DEG C of denaturation, 15s, 60 DEG C, 30-45s of annealing/extension, common 30-40 circulation.

LncRNA-TMP125 in qRT-PCR detection Bladder Cancer samples, starting point represent the logarithmic phase of product accumulation Start, the fluorescence signal of the product is exponentially increased, as Ct values.It can predict that the starting of target gene product is dense according to Ct values Degree, i.e., in the case of PCR reaction condition identicals, target gene initial concentration is bigger, then Ct values are lower.

Cut- using the upper bound (X ± 2.79SD) of 95% credibility interval of control class mean by cancer as this diagnostic test Off values, its value are 4.206.Under the conditions of this cut off value, the sensitivity of this diagnostic test is 92%, specificity 83%.Specifically It is as follows：

Clinical Sensitivity, which can be used to weigh certain testing inspection, to be gone out to have the ability of patient, and sensitivity is by actual ill people Correctly it is determined as the ratio of true positives, this experiment sensitivity=89/ (89+11) × %=89%.

Clinical specificity is the ability that balancing tests correctly judge no patient, and specificity is that actual disease-free people is correct Ground is determined as the ratio of true negative.This experiment specificity=87/ (13+87) × %=87%.The detection kit of the present invention is used In the detection of Bladder Cancer, there are higher sensitivity and specificity.

Although above with general explanation and specific embodiment, the present invention is described in detail, at this On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore, These modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.

Sequence table

<110>Lee's Chong

<120>A kind of kit that carcinoma of urinary bladder is detected using long-chain non-coding RNA

<130> 201710

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1122

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 1

tgtctttcct tctactgcac actgagctcc tgaaggcact ggggcttgct cccatttgta 60

ccctggagca cacagtaggt gcttactaag tgactgtgga gcagaacttc taatattttt 120

gaaacacaga tttggaaaca tcttcccaga aaacagacta caacaaatca tttccctggt 180

atgaaggaat gaagtttttt gtttctttca tagtcctaaa aaaatccccc aagagctcac 240

cagcatgaaa gcactagctc tgaatatgat atggaggaac aatgatttga agaccttcga 300

aaggcaagca acttgacacg gggcatcttt ctagatcatt gtttggaaga gaaccatcag 360

gacgatccca ggggtcctgt gaaacgctgt tggcctgggc aaggagggaa ctggtgctca 420

tcactggccc tcttcagaaa caatctgctc cgtccaagtg caatatgagt atttccagct 480

gaccatgggg aaacttgttt tctagtttat cccagctaca ttatcaagaa aatagtcaat 540

ggagtcaaca aatgcaaacc tctggaaaac atttcagaga ctggcttgtt gctacaacaa 600

gaacggtgat atcataatca ccattgtgga gcagatgcca ggtagcattg ctaacctctt 660

cagaataatt gacatttttc taacatgcag agtggccata acaaccactt cagctacaca 720

atagcctcca ccgtaacact tccttagcaa cccgcttaac accgtgactt tgaaatcatc 780

cgctattttg gcagcaggat gaatgtttgc tggtcgtgaa cttttcaaag gtcatcagct 840

ggaaacaagc ctgcccactt cctttgccta ccccgccact gcctgtcgta cccatccatc 900

acaggacata atgcttaagc acttgaggaa aagataatgc accgcttaca ttttcctagt 960

ggaacaataa attttatttc ggggatgttg ggggaaaaaa gtcgtagtgt tactgaccca 1020

tgttttatta acctgtctcc aattaacaga aaggatgaca ctaattggaa acacaccatt 1080

ttgcttaagc aattaatctt ttttggttta tcttttaaag cc 1122

<210> 2

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 2

caccagcatg aaagcactag 20

<210> 3

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

aaatactcat attgcacttg 20

<210> 4

<211> 30

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

caggacgatc ccaggggtcc tgtgaaacgc 30

Claims

A kind of 1. kit that carcinoma of urinary bladder is detected using long-chain non-coding RNA, it is characterised in that

The kit includes：

The anti-sense primer as described in sense primer and SEQ ID No.3 as described in SEQ ID No.2；

Fluorescent marker；

Include the standard DNA template of nucleotide sequence described in SEQ ID No.1；

PCR reaction solutions.
2. the kit according to claim 1 that carcinoma of urinary bladder is detected using long-chain non-coding RNA, it is characterised in that

The PCR reaction solutions include 2 × Super TaqMan Onestep Buffer, Super Onestep Enzyme, visit Pin, RNase-Free Water.
3. the kit according to claim 2 that carcinoma of urinary bladder is detected using long-chain non-coding RNA, it is characterised in that

PCR reaction systems are 25ul reaction systems, wherein, 2xSuper TaqMan Onestep Buffer 12.5ul；Upstream Primer, 10uM, 0.5ul；Anti-sense primer, 10uM, 0.5ul；Probe, 10pM, 0.5ul；Super Onestep Enzyme, 1.0ul；RNA Template, 10pg-100ng.
4. the kit according to claim 1 that carcinoma of urinary bladder is detected using long-chain non-coding RNA, it is characterised in that

The standard form DNA is transformed into DH5 α competent cells, for the matter containing sequence shown in SEQ ID No.1 of extraction Grain.
5. the kit according to claim 1 that carcinoma of urinary bladder is detected using long-chain non-coding RNA, it is characterised in that

The fluorescent marker is SYBR Green, TaqMan probe, molecular beacon, double cross probe or combined probe.
6. the kit according to claim 5 that carcinoma of urinary bladder is detected using long-chain non-coding RNA, it is characterised in that

The TaqMan probe sequence is as shown in SEQ ID No.4.
7. the kit according to claim 1 that carcinoma of urinary bladder is detected using long-chain non-coding RNA, it is characterised in that

The kit also includes RNA extracts reagent, and the extracts reagent of the RNA is Thermo Fisher Scientific The TRIzol reagents of company.
8. the kit according to claim 1 that carcinoma of urinary bladder is detected using long-chain non-coding RNA, it is characterised in that

The kit also includes nucleic acid quantification instrument.
9. the kit according to claim 1 that carcinoma of urinary bladder is detected using long-chain non-coding RNA, it is characterised in that

The kit also includes fluorescent quantitation amplification instrument.