CN103923984A - Development and application of esophageal squamous carcinoma detection kit - Google Patents
Development and application of esophageal squamous carcinoma detection kit Download PDFInfo
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Abstract
The invention discloses development and application of an esophageal squamous carcinoma detection kit. The invention relates to a long-chain non-coding RNA and application thereof. Specific real-time quantitative PCR (Polymerase Chain Reaction) primers and a probe are designed and synthesized according to a sequence of the long-chain non-coding RNA, and a preparation for auxiliary diagnosis or therapeutic effect predication of esophageal squamous carcinoma is prepared. The expression level of the long-chain non-coding RNA is detected in a specimen of an esophageal squamous carcinoma clinical case by using a specific real-time quantitative PCR preparation, remarkable up regulation of the long-chain non-coding RNA in the expression of the specific real-time quantitative PCR is discovered, and the expression level is highly correlated to the clinical stages of a patient suffering from the esophageal squamous carcinoma; the specific detection sequence related in development and application disclosed by the invention is expected to prepare the preparation for auxiliary diagnosis or therapeutic effect predication or prognosis of the esophagus cancer.
Description
Technical field
The invention belongs to oncomolecularbiology field, be specifically related to a kind of long-chain non-coding RNA and application thereof, particularly, the present invention relates to the application of a kind of long-chain non-coding RNA in preparation esophageal carcinoma auxiliary diagnosis or prognosis preparation.
Background technology
The esophageal carcinoma (Esophageal Carcinoma, EC) is the malignant disease of serious harm universe health, and in global range, its M & M occupies respectively the 8th and the 6th.EC mainly contains two kinds of histological type: esophageal squamous cell carcinoma (Esophageal Squamous Cell Carcinoma, ESCC) and adenocarcinoma of esophagus (Esophageal Adenocarcinoma, EAC), China's histological type be take squama cancer as main (accounting for more than 90%).The latest information demonstration of announcing according to the World Health Organization, the annual new ESCC patient of China surpasses 250,000 people, and because of patient approximately 200,000 people of death from esophageal carcinoma, sickness rate occupies the 5th of national all kinds of malignant tumour, and mortality ratio occupies the 4th, all far super world average level.Although, for the technique means of esophageal carcinoma inspection and treatment, to update clinically at present, overall 5 years survival rates of patient are still extremely low.Since nearly half a century, about the pathogenetic research of EC, in mRNA, protein and miRNA field, obtained a series of achievements both at home and abroad, but its definite pathogenesis is at present still among positive research and probe.(Jemal A, et al.Global cancer statistics.CA:a cancer joumal for clinicians2011,61:69-90; Enzinger PC, et al.Esophageal cancer.N Engl J Med2003,349:2241-2252; Old ten thousand blue or green .2004-2005 China's Incidences and dead estimation. Chinese Journal of Oncology 2009,31:664-668; He Jie, etc. esophageal cancer in China present epidemiology, diagnosis and treatment present situation and future countermeasure. Cancer in China magazine, 2011, (7): 501-504.)
After human genome order-checking plan completes, confirm that the protein coding gene receiving much concern for a long time only accounts for whole 2% of the group of transcribing, the transcription product over 98% is ncRNA (noncoding RNA, ncRNA).Along with RNA group is learned progress of research, originally be considered to genome and transcribe the ncRNA of " noise ", progressively confirmed that it is comprising many species of the mankind, or even in the physiology of plant and pathology activity, all there is extensive and diversified function rarely known by the people.Confirm that at present the ncRNAs with regulating and controlling effect is mainly divided into the LncRNAs of length >200nt and the microRNAs of <200nt, although microRNAs studies the relatively deep little ncRNAs of a class so far, but it is found that subsequently in each species, LncRNAs not only quantity is large, kind is many, and transcribing and translating at gene, cytodifferentiation and ontogeny, regulating and controlling effect in the vital movements such as heredity and epigenetic is more extensive compared with microRNAs, meticulous and complicated (Bimey E, et al.Identification and analysis of functional elements in1%of the human genome by the ENCODE pilot project.Nature2007, 447:799-816, Wilusz JE, et al.Long noncoding RNAs:Functional smprises from the RNA world.Genes and Development2009,23:1494-1504, Prasanth KV, et al.Eukaryotic regulatory RNAs:An answer to the ' genome complexityconundrum.Genes and Development2007,21:11-42, Tsai MC.et al.Long intergenic noncoding RNAs:new links in cancer progression.Cancer Res2011,71:3-7, Pauli A, et al.Non-coding RNAs regulators of embryogenesis.Nature Reviews Genetics2011,12:136-149, Van LeeuwenS, et al.Long non-coding RNAs:Guardians of development.Differentiation2010,80:175-183,
uA, et al.Long Noncoding RNAs with Enhancer-like Function in Human Cells.Cell2010.143:46-58, Caley DP, et al.Long noncoding RNAs, chromatin, and development.The Scientific World Journal2010.10:90-102.).
Long-chain non-coding RNA (long non-coding RNA, lncRNA) be the non-coding RNA that a class transcript length surpasses 200 Nucleotide, research in recent years finds that it is that a class has the RNA that important biomolecule is learned function, participate in genomic imprinting, karyomit(e) silence, chromatin modification, transcriptional activation, transcribe interference, the multiple important regulation process such as the interior transportation of core, in the vital movements such as cytodifferentiation and growth, genetic transcription and translation, heredity and epigenetic, all bring into play important regulating and controlling effect.In recent years, increasing authority studies confirm that lncRNA plays a part to suppress or promote tumour in the developing of tumour, and at aspects such as modulate tumor cell proliferation, apoptosis, cell cycle, Invasion and Metastasis abilities, all has very vital role.At present existing more LncRNAs is proved in the mankind's kinds of tumors that comprises mammary cancer, prostate cancer, melanoma, liver cancer, colorectal cancer, bladder cancer etc. and there are differences and express and carry out important adjusting function (Gupta RA, et al.Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis.Nature2010,464:1071-1076; Cui Z.et al.The prostate cancer-up-regulated long noncoding RNA PlncRNA-1modulates apoptosis and proliferation through reciprocal regulation of androgen receptor.Urologic Oncology:Seminars and Original Investigations2013,3l:1117-1123; Khaitan D.et al.The melanoma-upregulated long noncoding RNA SPRY4-IT1modulates apoptosis and invasion.Cancer Research2011.71:3852-3862; Du Y, et al.Elevation of Highly Up-regulated in Liver Cancer (HULC) bv Hepatitis B Virus X Protein Promotes Hepatoma Cell Proliferation via Down-regulating p18.J Biol Chem2012,287:26302-26311; Ling H, et al.CCAT2, a novel noncoding RNA mapping to8q24, underlies metastatic progression and chromosomal instability in colon cancer.Genome Research2013,23:1446-1461; Liu Z.et al.Downregulation of GAS5Promotes Bladder Cancer Cell Proliferation, Partly by Regulating CDK6.PLoS ONE2013,8:9Article Number e73991.).For exploring LncRNA, in ESCC, whether there is function, contriver place seminar is in people ESCC tissue and clone, with the current verified LncRNA in other tumour with correlation function, carry out exploratory study, the LncRNA HOTAIR that confirmation is found in breast cancer tissue is differential expression in ESCC cancer and healthy tissues not only, and in ESCC cell strain obvious inhibition tumor cell apoptosis promote metastases (Chen FJ, et al.Upregulation of the long non-coding rna hotair promotes esophageal squamous cell carcinoma metastasis and poor prognosis.Molecular Carcinogenesis2013, 52:908-915.).In addition, contriver also confirms that the PlncRNA-1 finding in prostate gland all significantly raises and can regulate and control tumor cell proliferation ability (Wang CM in ESCC tissue, et al.Upregulation of the Long Non-coding RNA PlncRNA-1Promotes Esophageal Squamous Carcinoma Cell Proliferation and Correlates with Advanced Clinical Stage, Dig Dis Sci2013.doi:10.1007/s10620-013-2956-7).
Yet, follow the trail of document and find except the seminar of contriver place, at present also less about the lncRNA express spectra of esophageal squamous cell carcinoma and functional research of index of correlation thereof.LncRNA express spectra for system exploration ESCC, find, to ESCC, one group of lncRNAs that development specificity is relevant occurs, contriver adopts chip technology to screen the lncRNA of a remarkable high expression level in people ESCC cancerous tissue, this gene is named as RP11-697M17.1 (Ensemble database), and it is transcribed region and is positioned at karyomit(e) antisense strand 186,435 No. 8,038-186,478,330bp, full length gene is about 1164bp.Contriver studies confirm that: compare with healthy tissues, this lncRNA is remarkable high expression level in people ESCC cancerous tissue, and in organizing qRT-PCR (Quantitative Real-time Polymerase Chain Reaction, real-time quantitative PCR) experiment, the large sample in later stage further confirms that the expression of this index in people ESCC cancerous tissue is significantly higher than healthy tissues.For ease of later stage research and discussion, contriver is by its called after HUESCC-lncRNA2 (highly up-regulated in esophageal squamous cell carcinoma, long noncoding RNA2).The inventor has paid close attention to the lncRNA express spectra of ESCC, the novel gene regulating factor of this class will be expected to the further abundant and perfect research that comprises the Tumorigenesis of esophageal squamous cell carcinoma, also for finding that diagnosing tumor and the mark of prognosis judgement, new oncotherapy target spot bring hope.
Summary of the invention
LncRNA express spectra for systematic study people esophageal squamous cell carcinoma, find to occur and the closely-related new lncRNAs of development with people's esophageal squamous cell carcinoma, the 6 pairs of esophageal carcinoma being provided by contriver and healthy tissues sample, RNA by Qiagen company extracts test kit (RNeasy Micro Kit, article No. 74004) extract after RNA, adopt the brilliant core lncRNA chip V2 (4 * 180K) of Beijing Boao Biological Co., Ltd to detect the lncRNA that filters out a remarkable high expression level in human esophageal carcinoma, called after HUESCC-lncRNA2, its gene order is as shown in sequence table SEQ ID NO.1.Later stage is found in 85 pairs of samples through the totally 110 pairs of people ESCC cancers and healthy tissues sample qRT-PCR checking that this lncRNA expresses in tumor tissues and significantly raises.This lncRNA is expected to become the mark of diagnosing tumor and prognosis judgement, and the while is also for the treatment of tumour provides new target spot.
The lncRNA sequence and the application method thereof that the object of this invention is to provide a kind of detection high expression level in esophageal carcinoma tissue.
The lncRNA express spectra of systematic research esophageal squamous cell carcinoma of the present invention, finds to occur and the closely-related new lncRNA of development with esophageal squamous cell carcinoma.
The HUESCC-lncRNA2 sequence that the object of this invention is to provide a kind of detection high expression level in esophageal carcinoma tissue.
The invention provides the detection application method of described HUESCC-lncRNA2 sequence, the preparation according to this sequence for the preparation of esophageal carcinoma auxiliary diagnosis or outcome prediction.
The present invention according to described HUESCC-lncRNA2 sequences Design 3 pairs of primers for detection of HUESCC-lncRNA2 sequence.
Pairl upstream primer: SEQ ID NO.2
Pairl downstream primer: SEQ ID NO.3
Pair2 upstream primer: SEQ ID NO.4
Pair2 downstream primer: SEQ ID NO.5
Pair3 upstream primer: SEQ ID NO.6
Pair3 downstream primer: SEQ ID NO.7
The present invention is according to described HUESCC-lncRNA2 sequences Design and synthesize the detection primer sets for real-time quantitative PCR.Described primer sets is applicable to the detection of SYBR Green, TaqMan probe, molecular beacon, double cross probe, combined probe etc.
The primer sets preferably detecting for dye class real-time quantitative PCR is respectively:
Upstream primer: SEQ ID NO.4
Downstream primer: SEQ ID NO.5
In the present invention, qRT-PCR, real-time quantitative PCR, quantitative fluorescent PCR are identical concepts, can mutually replace use.
Another object of the present invention is to provide a kind of PCR kit for fluorescence quantitative and using method of the HUESCC-lncRNA2 of detection expression level, this PCR kit for fluorescence quantitative is suitable for existing at present all types fluorescence quantitative gene extender on market, highly sensitive, quantitatively quick and precisely, good stability, have a good application prospect.
The present invention has prepared a kind of dye class real-time quantitative PCR test kit of the lncRNA of detection expression level, and component is as follows: Auele Specific Primer, standard DNA template, fluorescence dye, real-time quantitative PCR reaction solution.Wherein said Auele Specific Primer comprises upstream primer and downstream primer, and upstream primer sequence is SEQ IDNO.4, and downstream primer sequence is SEQ ID NO.5.Described fluorescence quantitative PCR reaction solution comprises dNTP, Mg
2+, Taq enzyme and buffer damping fluid.The preferred SYBR Green of described fluorescence dye II, the preferred warm start enzyme of Taq enzyme.
The invention also discloses a kind of using method that detects the dye class PCR kit for fluorescence quantitative of esophageal squamous cell carcinoma, quantitative fluorescent PCR system:
Upstream primer (10 μ mol/L) 2 μ L; Downstream primer (10 μ tmol/L) 2 μ L; Sample cDNA4 μ L (or standard DNA template DNA 2 μ L); 50 * ROX Reference Dye1 μ L; 2 * SYBR PremexExTaq II (TIi RNaseH Plus), 25 μ L, add ionized water to 50 μ L.Quantitative fluorescent PCR program: 95 ℃ of 30s denaturations, connect 40 circulations: 95 ℃ of 5s, 60 ℃ of 30-34s.
The invention also discloses a kind of detection method of long-chain non-coding RNA, comprise extraction, the preparation of sample cDNA, the amplification of HUESCC-lncRNA2 of the total RNA of sample.
The concrete steps of the extraction of the total RNA of above-described sample, the preparation of sample cDNA, HUESCC-lncRNA2 amplification comprise:
1) extraction of the total RNA of sample: according to Life Technologies company
reagent (article No. 15596026) required reagent and step are extracted the Total RNA of esophageal carcinoma tissue or tumour; Purity and the concentration of using the NanoDrop ND-1000 nucleic acid quantification instrument RNA that quantitatively (NanoDrop Technologies, Wilmington, Delaware) is quantitative extracted, the integrity of the RNA of extraction is guaranteed in the quality inspection of denaturing formaldehyde gel electrophoresis again.
2) preparation of sample cDNA: adopt TaKaRa test kit PrimeScript
tMrT reagent Kit with gDNA Eraser (Perfect Real Time) (article No. RR047A) is to the synthetic cDNA of total RNA reverse transcription of extracting; This test kit includes RNase-Free DNase, can effectively remove the genomic dna mixing.
The first step is removed genomic dna reaction, and reaction system and condition are as follows:
| Reagent | Usage quantity |
| 5×gDNA?Eraser?Buffer | 2.0μl |
| gDNA?Eraser | 1.0μl |
| Total?RNA | 1μg |
| RNase?Free?dH2O | Add water to 10 μ l |
Above-mentioned component is mixed to rear 42 ℃ of reaction 2min;
Second step reverse transcription reaction, reaction system and condition are as follows:
| Reagent | Usage quantity |
| The reaction solution of step 1 | 10.0μl |
| PrimeScript?RT?Enzyme?Mix?I | 1.0μl |
| ITPrimer?Mix | 1.0μl |
| 5×PrimeScript?Buffer2 | 4μl |
| RNase?Free?dH 2O | 4.0μl |
| Cumulative volume | 20μl |
Above-mentioned component is mixed to latter 37 ℃ and hatch 15min, then 85 ℃ of deactivation 5sec, obtain cDNA.
3) amplification of HUESCC-lncRNA2: adopt TAKARA SYBR Premix Ex Tr
tMiI (TIi RNaseH Plus) fluorescence quantitative kit, the cDNA of reverse transcription of take carries out fluorescent quantitative PCR as template.
Dye class quantitative fluorescent PCR system: upstream primer (10 μ mol/L) 2 μ L; Downstream primer (10 μ mol/L) 2 μ L; Sample cDNA4 μ L (or standard DNA template DNA 2 μ L); 50 * ROX Reference Dye (or 50 * ROX Reference Dye II), 1 μ L; 2 * SYBR Premex Ex Taq II (TIi RNaseH Plus), 25 μ L, add ionized water to 50 μ L.Quantitative fluorescent PCR program: 95 ℃ of 30s denaturations, connect 40 circulations: 95 ℃ of 5s, 60 ℃ of 30-34s.
The present invention has also detected test kit susceptibility, and result shows that test kit sensing range of the present invention is 10
7-10
2copies/ μ L, minimum concentrations is 100copies/ μ L.
By the detection to positive, find that dye class fluorescence quantitative kit Detection accuracy of the present invention is 73-79%, continuous repetition for 3 times tested, and experimental result is stable.
Accompanying drawing explanation
Fig. 1 .LncRNA chip dendrogram
The LncRNA chip cluster that shows 6 pairs of esophageal squamous cell carcinoma Ai Yu cancer beside organism differential expressions
Fig. 2. Auele Specific Primer the selection result figure
Effect for row agarose gel electrophoresis test primer after 3 pairs of Auele Specific Primer pcr amplifications of the sequences Design of this LncRNA
Fig. 3. the preliminary qRT-PCR detected result (2 of HUESCC-lncRNA2 of first 30 routine ESCC clinical samples
-Δ ctmapping)
Fig. 4. the HUESCC-lncRNA2 of the routine ESCC clinical sample of second batch 80 verifies qRT-PCR detected result (2 again
-Δ cdmapping)
Fig. 5. the two batches of totally 110 routine sample HUESCC-lncRNA2 Ai Yu differential expression qRT-PCR of cancer beside organism detection statistics analyses (2
-Δ Δ ctvalue compares,
*p<0.05,
*p<0.01)
Fig. 6 .qRT-PCR detects expression and the patient clinical correlation analysis (2 by stages of HUESCC-lncRNA2
-Δ Δ ctvalue compares,
*p<0.05,
*p<0.01)
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention, only for explaining the present invention, and can not be interpreted as limitation of the present invention.Those having ordinary skill in the art will appreciate that: in the situation that not departing from principle of the present invention and aim, can carry out multiple variation, modification, replacement and modification to these embodiment, scope of the present invention is limited by claim and equivalent thereof.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition or the condition examinations of advising according to manufacturer.
The IncRNA chip expression analysis of embodiment 1 people's esophageal squamous cell carcinoma cancer and healthy tissues
One materials and methods
1, material
Tissue samples comes from inpatient's excision sample of 6 pairs of patients with esophageal squamous cells, every pair of normal esophageal tissue that comprises esophageal neoplasm tissue and pairing.
2, method
The extraction of 2.1 tumor tissues and the total RNA of healthy tissues
The RNA that presses Qiagen company extracts test kit (RNeasy Micro Kit, article No. 74004) specification sheets extracts total RNA of Esophagus squamous cancer patient's tumor tissues and healthy tissues, this test kit includes RNase-Free DNase I (lyophilized), can effectively remove the genomic dna mixing.Purity and the concentration of using the NanoDrop ND-1000 nucleic acid quantification instrument RNA that quantitatively (NanoDrop Technologies, Wilmington, Delaware) is quantitative extracted, the integrity of the RNA of extraction is guaranteed in the quality inspection of denaturing formaldehyde gel electrophoresis again.
2.2 couples of sample RNA carry out fluorescent mark (brilliant core
cRNA amplification label test kit, catalog number: 360060-10)
2.2.1 the first chain cDNA is synthesized in reverse transcription
Take Total RNA as initial, and the T7Oligo that contains T7 promoter sequence (dT) Primer is primer, uses CbcScript enzymic synthesis the first chain cDNA.
2.2.2 synthesize 2
nd-strand cDNA
With RNase H, the RNA in heterozygosis chain is cut into short-movie section, it is primer extension that DNA Polymerase be take RNA short-movie section, synthetic 2
nd-strand cDNA, and the double-stranded cDNA of purifying.
2.2.3 in-vitro transcription is synthesized cRNA
Take cDNA as template, utilize T7Enzyme Mix to synthesize cRNA; Then use RNA Clean-up Kit (MN) purifying.
2.2.4 random primer reverse transcription
Get 5ug cRNA, with CbcScript II enzyme, Random Prime carries out reverse transcription, PCR NucleoSpinExtract II Kit (MN) purifying for reverse transcription product.
2.2.5cDNA use KLENOW enzyme labelling
Get above-mentioned reverse transcription product, the Random Primer of take carries out KLENOW enzyme labelling as primer, and PCR NucleoSpinExtract II Kit (MN) purifying for marked product, drains after purifying.(Cy5-dCTP?or?Cy3-dCTP(GE?Healthcare)。
2.3 hybridization and cleaning
The DNA of mark is dissolved in (2 * GEx Hyb Buffer (HI-RPM), 25% methane amide) in hybridization solution, in 45 ℃ of hybridization, spends the night.After hybridization finishes, first 42 ℃ of left and right, contain 0.2%SDS, in the liquid of 2 * SSC, wash 5min, then in 0.2 * SSC, room temperature is washed 5min.Slide can be used for scanning after drying.
2.4 chip scanning
Chip scans with Agilent G2565CA Microarray Scanner, obtains hybridizing picture.
The collection of 2.5 chip images and data analysis
2.5.1 fluorescent signal value is extracted
Adopt Feature Extraction image analysis software to analyze chip image, picture signal is converted into numerary signal.
2.5.2 differential gene screening
Raw data is input in GeneSpring GX software, adopts percentile shift method to be normalized signal value.Then use Absolute Fold change >=2, while Flag is labeled as the standard of Detected and carries out differential gene screening.
Two results
Fig. 1 is shown in LncRNA chip cluster analysis about people's esophageal squamous cell carcinoma.The lncRNAs of many up-regulateds and down-regulated expression is found in chip examination.Wherein HUESCC-lncRNA2 demonstrates in cancerous tissue and expresses significantly and raise, in view of it may exist specific expressedly in the cancerous tissue of people's esophageal squamous cell carcinoma, the present invention adopts extensive sample to carry out in batches the repeated authentication of index by following examples.
Embodiment 2qRT-PCR preliminary identification HUESCC-IncRNA2 is at the cancerous tissue of esophageal squamous cell carcinoma and the differential expression in healthy tissues
One, experiment material
Choose again other 30 to the cancerous tissue of (sample that is different from chip testing) people's esophageal squamous cell carcinoma and pairing healthy tissues, the differential expression of HUESCC-lncRNA2 is carried out to qRT-PCR preliminary identification.
Two, experimental technique and result
1 primer specificity is identified
The screening of 1.1 Auele Specific Primers
(1) from Ensemble database, extract the relevant transcript sequence of HUESCC-lncRNA2, and use according to the sequence of transcript by design of primers instrument (Primer-BLAST) the design primer of NCBI;
(2) primer after design is evaluated with Oligo7,3 pairs of primers of every design;
Pairl upstream primer: SEQ ID NO.2
Pairl downstream primer: SEQ ID NO.3
Pair2 upstream primer: SEQ ID NO.4
Pair2 downstream primer: SEQ ID NO.5
Pair3 upstream primer: SEQ ID NO.6
Pair3 downstream primer: SEQ ID NO.7
Through RT-PCR and agarose gel electrophoresis experimental verification, the primer sets following (Fig. 2) that screening detects for dye class real-time quantitative PCR:
Pair2 upstream primer: SEQ ID NO.4
Pair2 downstream primer: SEQ ID NO.5
(3) by the cancer of people's esophageal squamous cell carcinoma and healthy tissues according to Life Technologies company
reagent (article No. 15596026) required reagent and step are extracted total RNA; Purity and the concentration of using the NanoDrop ND-1000 nucleic acid quantification instrument RNA that quantitatively (NanoDrop Technologies, Wilmington, Delaware) is quantitative extracted, the integrity of the RNA of extraction is guaranteed in the quality inspection of denaturing formaldehyde gel electrophoresis again.
(4) adopt TaKaRa test kit PrimeScript
tMrT reagent Kit with gDNA Eraser (Perfect Real Time) (article No. RR047A) is to the synthetic cDNA of total RNA reverse transcription of extracting, and this test kit includes gDNA Eraser DNase, can effectively remove the genomic dna mixing.
The first step is removed genomic dna reaction, and reaction system and condition are as follows:
| Reagent | Usage quantity |
| 5×gDNA?Eraser?Buffer | 2.0μl |
| gDNA?Eraser | 1.0μl |
| Total?RNA | 1μg |
| RNase?Free?dH2O | Add water to 10 μ l |
Above-mentioned component is mixed to rear 42 ℃ of reaction 2min;
Second step reverse transcription reaction, reaction system and condition are as follows:
| Reagent | Usage quantity |
| The reaction solution of step 1 | 10.0μl |
| PrimeScript?RT?Enzyme?Mix?I | 1.0μl |
| RTPrimer?Mix | 1.0μl |
| 5×PrimeScript?Buffer2 | 4μl |
| RNase?Free?dH 2O | 4.0μl |
| Cumulative volume | 20μl |
Above-mentioned component is mixed to latter 37 ℃ and hatch 15min, then 85 ℃ of deactivation 5sec, obtain cDNA.The cDNA synthesizing of take is template, the negative control group of setting reaction groups and not adding cDNA template for the 3 pairs of primers respectively, with except primer dimer may, then the primer designing by step (2) carries out PCR reaction;
(5) electrophoresis detection, selects Marker DL1000 (TaKaRa).Primer selection standard: a. carries out entry evaluation by Marker to amplified fragments size, and amplified fragments size is identical with expection; B. amplified production only has one, guarantees the specificity of primer amplification.As shown in Figure 2, best primer pair is Pair2 to result, the Auele Specific Primer of upstream, and its sequence is shown in sequence table SEQ ID NO.4, the Auele Specific Primer in downstream, its sequence is shown in sequence table SEQ ID NO.5.
The extraction of the total RNA of 2 sample:
Adopt liquid nitrogen grinding method, according to Life Technologies company
reagent (article No. 15596026) required reagent and step are extracted the Total RNA of esophageal carcinoma tissue or tumour.Main operational steps is as follows:
(1) sample is chilled in rapidly in liquid nitrogen after in vitro, and the mortar of during extracting RNA, tissue being put into precooling grinds, and grinding limit, limit adds liquid nitrogen, and whole process does not all make liquid nitrogen volatilization dry;
(2) after tissue sample is ground into powder, when liquid nitrogen is evaporated completely substantially, in each mortar, add 1-2ml TRIZOL reagent, after adding, TRIZOL can be frozen into solid state, can continue to grind this solid powdered, along with mortar temperature recovery is to room temperature, the TRIZOL of solid state is progressively returned to liquid state, this TRIZOL agent transfer in glass homogenizer, more further homogenate 3-5min;
(3) TRIZOL agent transfer in 1.5ml centrifuge tube, the TRIZOL reagent of every 1 milliliter adds 0.2 milliliter of chloroform.Carefully build sample hose.With hand, shake energetically pipe 15 seconds, hatch 2-3 minute for 15-30 ℃.2-8 ℃, is no more than 12000g centrifugal 15 minutes.After centrifugal, mixture separation is the colourless water on red lower floor (phenol-chloroform phase), middle phase and upper strata, and RNA is present in water;
(4) water is transferred to a new pipe, mixes Virahol with precipitated rna from water, and every 1 milliliter of TRIZOL reagent for initial homogeneity is used 0.5 milliliter of Virahol, hatches sample 10 minutes for 15-30 ℃, and 2-8 ℃, is no more than 12000g centrifugal 10 minutes;
(5) abandon supernatant, once, every milliliter of TRIZOL reagent for initial homogeneity is used the ethanol of at least 1 milliliter 75% to the washing with alcohol RNA precipitation with 75%, vortex mixed, and 2-8 ℃, is no more than 7500g centrifugal 5 minutes;
(6) abandon supernatant, simple dry RNA precipitation (dry air or vacuum-drying 5-10 minute), dissolves several times RNA and is stored in-70 ℃ without the water piping and druming of RNA enzyme with 20-100ul, in this process, should be noted, this does not allow RNA precipitate complete drying, because will reduce its solubleness greatly.
The preparation of 3 standard DNA templates
To specifications, from the cancer and pairing healthy tissues of esophageal squamous cell carcinoma, utilize Invitrogen RTIZOL test kit to extract total RNA, and further adopt
rNA clean-up test kit (MACHEREY-NAGEL, Germany) total RNA was carried out to column purification, then carry out reverse transcription reaction, reverse transcription system is: reverse transcription random primer (2lamol/L) 1 μ L, total RNA4 μ L, dNTP mixture (every kind of 2.5mmol/L) 4 μ L, 0.1mol/L DTT2 μ L, SuperScript RNase H reversed transcriptive enzyme (200U/ μ L) 2 μ L (TAKARA) reaction conditionss are: 37 ℃ of water-bath 60min, 95 ℃ of 3min.
The cDNA that reverse transcription reaction is obtained carries out conventional PCR, reaction system and condition are as follows: 10 * Ex Taq buffer10 μ L, dNTP Mixture (each 2.5mmol/L) 4 μ L, Sequence NO.4 (10pmol) 4 μ L, Sequence NO.5 (10pmol) 4 μ L, eDNA (0.1-2 μ g) 5 μ L, Ex Taq archaeal dna polymerase 0.5 μ L, distilled water polishing is to 100uL.Reaction conditions is 94 ℃ of denaturation 5min; 94 ℃ of sex change 50s, 50 ℃ of annealing 50s, 72 ℃ are extended 50s, 35cycles; Last 72 ℃ are extended 10min.
Sample 5 μ L, the product of pcr amplification is carried out to agarose gel electrophoresis detection, cut glue and reclaim also purifying (recovery use test kit: EZ-10Spin Column DNA Gel Extraction Kit), purified product is connected to pGM-T cloning vector, is transformed into subsequently in DH5a competent cell.It by sequence, is the Auele Specific Primer screening positive clone of SEQ ID NO.4 and SEQ ID NO.5.After positive colony amplification, extract plasmid DNA, plasmid DNA adopts quantitatively (NanoDrop Technologies of NanoDrop ND-1000 nucleic acid quantification instrument, Wilmington, Delaware) and for the preparation of typical curve, (standard DNA template concentrations scope is 10 as standard substance to do 10 times of serial dilutions
8-10
2copies/ μ l).
4 sensitivity experiments
Getting that recombinant plasmid dilutes is in proportion 10
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2individual copy/μ L, carries out quantitative fluorescent PCR, the detection sensitivity that the minimum concentration of test positive of take is the method.The method sensing range that this institute sets up is 10
8-10
2copies/ μ L, minimum concentrations is 100copies/ μ L.
5 synthetic cDNA templates
Get total RNA of above-mentioned 30 pairs of esophageal squamous cell carcinoma cancerous tissues and healthy tissues, adopt TaKaRa test kit PrimeScript
tMrT reagent Kit with gDNA Eraser (Perfect Real Time) (article No. RR047A) is to the synthetic cDNA of total RNA reverse transcription of extracting, and this test kit includes gDNA Eraser DNase, can effectively remove the genomic dna mixing.
The first step is removed genomic dna reaction, and reaction system and condition are as follows:
| Reagent | Usage quantity |
| 5×gDNA?Eraser?Buffer | 2.0μl |
| gDNA?Eraser | 1.0μl |
| Total?RNA | 1μg |
| RNase?Free?dH2O | Add water to 10 μ l |
Above-mentioned component is mixed to rear 42 ℃ of reaction 2min;
Second step reverse transcription reaction, reaction system and condition are as follows:
| Reagent | Usage quantity |
| The reaction solution of step 1 | 10.0μl |
| PrimeScript?RT?Enzyme?Mix?I | 1.0μl |
| RT?Primer?Mix | 1.0μl |
| 5×PrimeScript?Buffer2 | 4μl |
| RNase?Free?dH 2O | 4.0μl |
| Cumulative volume | 20μl |
Above-mentioned component is mixed to latter 37 ℃ and hatch 15min, then 85 ℃ of deactivation 5sec, obtain cDNA.
6 dye class fluorescence quantitative PCR detection HUESCC-lncRNA2 expression amounts
Adopt TAKARA SYBR Premix Ex Taq
tMiI (TIi RNaseH Plus) fluorescence quantitative kit (article No. RR820A).50 μ LqRT-PCR reaction systems comprise: upstream primer (10 μ mol/L) 2 μ L; Downstream primer (10 μ mol/L) 2 μ L; Sample cDNA4 μ L; 50 * ROX Reference Dye1 μ L; 2 * SYBR Premex Ex Taq II (TIi RNaseH Plus), 25 μ L, add ionized water to 50 μ L.Instrument adopts Applied Biosystems7500.Quantitative fluorescent PCR program: 95 ℃ of 30s denaturations, connect 40 circulations: 95 ℃ of 5s, 60 ℃ of 30-34s.
According to the relative quantification formula of qRT-PCR: 2
-Δ Ctcalculate respectively the expression level of HUESCC-lncRNA2 in patients with esophageal squamous cell carcinoma cancerous tissue (T) and healthy tissues (N), comparative result is as shown in Figure 4: qRT-PCR stable amplification result, wherein the expression level of HUESCC-lncRNA2 in healthy tissues mainly concentrates on 0.000-0.001, in cancerous tissue, the expression amount of HUESCC-lncRNA2 is apparently higher than healthy tissues, mainly concentrate on 0.002-0.150, these results suggest that this index general high expression level in tumor tissues.According to relative expression quantity T-N>0, be defined as this index up-regulated again; T-N<0 is defined as this index down-regulated expression.This experimental result shows: HUESCC-lncRNA2 22 pairs of up-regulated expressions in 30 pairs of ESCC cancers and pairing healthy tissues, again according to formula: up-regulated expression number of cases/always detect the positive rate that number of cases x100% defines this index, the positive rate of this index is 73.33%.
Embodiment 3qRT-PCR further verifies that HUESCC-IncRNA2 is at the cancerous tissue of esophageal squamous cell carcinoma and the differential expression in healthy tissues
1, qRT-PCR test kit forms
1.1 dye class HUESCC-lncRNA2qRT-PCR test kits form:
(1) upstream primer: SEQ ID NO.4
(2) downstream primer: SEQ ID NO.5
Other reagent are with reference to SYBR Premix Ex Taq
tMiI (Tli RNaseH Plus) fluorescence quantitative kit (Code No.RR820A).
The detection of 2.HUESCC-lncRNA2qRT-PCR
The preparation of 2.1 total RNA
Choose other 80 couples of esophageal squamous cell carcinoma patients' cancerous tissue and pairing healthy tissues, according to Life Technologies company
reagent (article No. 15596026) required reagent and step are extracted total RNA, specifically referring to specification sheets.Purity and the concentration of using the NanoDrop ND-1000 nucleic acid quantification instrument RNA that quantitatively (NanoDrop Technologies, Wilmington, Delaware) is quantitative extracted, the integrity of the RNA of extraction is guaranteed in the quality inspection of denaturing formaldehyde gel electrophoresis again.
2.2cDNA synthetic
Adopt TaKaRa test kit PrimeScript
tMrT reagent Kit with gDNA Eraser (Perfect Real Time) (article No. RR047A), carries out reverse transcription reaction by detecting qualified total RNA.
2.3qRT-PCR detect
QRT-PCR adopts Applied Biosystems7500 at instrument.QRT-PCR response procedures is as fluorescence dye class real-time quantitative PCR detection HUESCC-lncRNA2 expression amount in embodiment 2.
3 detected results
Result as shown in Figure 4, adopt dye class real time quantitative PCR method, choose other 80 pairs of patients with esophageal squamous cell cancers and the checking of pairing healthy tissues enlarged sample amount, result demonstration HUESCC-lncRNA2 expresses in 63 routine specimens and obviously raises, and Positive rate is 79%.Above this index of result proving again is general high expression level in tumor tissues.We carry out 3 qRT-PCR checks of repetition to above-mentioned sample, and result repeatability reaches 100%, shows that the repeatability of test kit of the present invention and stability are better.
Embodiment 4HUESCC-IncRNA2 is in the potential value analysis of ESCC diagnosis and prognosis judgement
Detecting on the basis of HUESCC-lncRNA2 high expression level in ESCC tissue, clinical and pathological data in conjunction with patient, the expression of HUESCC-lncRNA2 and different pathological and the dependency between clinical stages (concrete with reference to new the 7th edition esophageal carcinoma TNM of UICC by stages standard) have further been analyzed, inquired into HUESCC-lncRNA2 in people's esophageal squamous cell carcinoma with the diagnosis of disease, comprise that it expresses the potential value that the aspects such as selection with the relation of clinical/pathological staging, prognosis judgement, treatment plan have.
QRT-PCR reaction result is used to SPSS For Windows20.0 software, related data is through Normal test check, according to data type, select Mann-Whitney check to carry out data analysis, P<0.05 thinks that the differential expression of this index has statistical significance.QRT-PCR interpretation of result HUESCC-lncRNA2 expression level in 110 pairs of ESCC samples is significantly higher than pairing normal esophageal tissue (Fig. 5, P<1 * 10
-6).In addition the expression of HUESCC-lncRNA2 and clinical stages height correlation (Fig. 6): the expression of HUESCC-lncRNA2 in II B-IIIC phase ESCC patient, is significantly higher than 0-IIA phase patient (P<0.05).Along with going deep into of later stage result of study, function and the mechanism of action thereof of HUESCC-lncRNA2 in ESCC will progressively be illustrated, HUESCC-lncRNA2 can not only become the biomarker that novel diagnosis is relevant, is more expected to become new ESCC treatment target spot to improve, to improve clinical ESCC result for the treatment of.Obviously, find more, effective as HUESCC-lncRNA2 and be expected to participate in auxiliary ESCC diagnosis, treatment and the relevant biomarker of prognosis more accurately, be of great practical significance.
Claims (8)
1. a long-chain non-coding RNA is in the application for the preparation of in esophageal carcinoma auxiliary diagnosis or curative effect predication reagent.
2. application according to claim 1, is characterized in that the described esophageal carcinoma is esophageal squamous cell carcinoma.
3. application according to claim 1, is characterized in that described long-chain non-coding RNA sequence is as shown in SEQ ID NO.1 in sequence table.
4. application according to claim 1, is characterized in that, the described reagent for esophageal carcinoma auxiliary diagnosis or outcome prediction is real-time quantitative PCR detection reagent.
5. the real time quantitative PCR detecting reagent kit for esophageal carcinoma auxiliary diagnosis or outcome prediction, it is characterized in that, comprise that the long-chain non-coding RNA of classifying SEQ ID NO.1 as according to nucleotides sequence designs and synthesizes out specificity for the detection primer of real-time quantitative PCR.
6. test kit according to claim 5, is characterized in that, the Auele Specific Primer detecting for long-chain non-coding RNA real-time quantitative PCR is as shown in sequence table SEQ ID NO.2 and SEQ ID NO.3.
7. a detection method for long-chain non-coding RNA, comprises extraction, the preparation of sample cDNA, the amplification of lncRNA of sample RNA.
8. detection method according to claim 7, is characterized in that, the extraction of described sample RNA, the preparation of sample cDNA, the concrete steps of the amplification of lncRNA comprise:
1) extraction of the total RNA of sample: according to Life Technologies company
reagent place needs reagent and step to extract the Total RNA of esophageal carcinoma tissue or tumour; Purity and the concentration of using the NanoDrop ND-1000 nucleic acid quantification instrument RNA that quantitatively (NanoDrop Technologies, Wilmington, Delaware) is quantitative extracted, the integrity of the RNA of extraction is guaranteed in the quality inspection of denaturing formaldehyde gel electrophoresis again.
2) preparation of sample cDNA: adopt TaKaRa test kit PrimeScript
tMrT reagent Kit with gDNA Eraser (Perfect Real Time) (article No. RR047A) is to the synthetic cDNA of total RNA reverse transcription of extracting; This test kit includes RNase-Free DNase, can effectively remove the genomic dna mixing.
3) amplification of lncRNA: adopt TAKARA SYBR Premix Ex Taq
tMiI (TIi RNaseH Plus) fluorescence quantitative kit, the cDNA of reverse transcription of take carries out fluorescent quantitative PCR as template.
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| CN110305961A (en) * | 2019-07-16 | 2019-10-08 | 南方医科大学深圳医院 | The application of miR-1207 and its target gene in detection larynx squamous carcinoma |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109593857A (en) * | 2019-01-31 | 2019-04-09 | 江苏万成生物医学研究院有限公司 | In blood circ-DLG1 molecular marker diagnosis esophageal squamous cell carcinoma by stages in application |
| CN109593858A (en) * | 2019-01-31 | 2019-04-09 | 江苏万成生物医学研究院有限公司 | Application of the circ-PLEKHA1 molecular marker in diagnosis esophageal squamous cell carcinoma in blood |
| CN110305961A (en) * | 2019-07-16 | 2019-10-08 | 南方医科大学深圳医院 | The application of miR-1207 and its target gene in detection larynx squamous carcinoma |
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