CN103952476B - A kind of detection and application of long-chain non-coding RNA - Google Patents
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Abstract
The present invention relates to a kind of long-chain non-coding RNA and its application, according to the long-chain non-coding RNA sequence, designs and synthesizes specific real-time quantitative PCR primer and probe, prepares the preparation for cancer of the esophagus auxiliary diagnosis or outcome prediction.Utilize real-time quantitative PCR preparation, the expression of the long-chain non-coding RNA is detected in cancer of the esophagus clinical case sample, it was found that the long-chain non-coding RNA is significantly lowered in Expression in Esophageal Carcinoma, the present invention is expected to prepare the preparation for cancer of the esophagus auxiliary diagnosis, outcome prediction or Index for diagnosis.
Description
Technical field
The invention belongs to oncomolecularbiology field, and in particular to a kind of long-chain non-coding RNA and its application, it is specific and
Speech, the present invention relates to a kind of application of long-chain non-coding RNA in cancer of the esophagus auxiliary diagnosis or prognosis preparation is prepared.
Background technology
The cancer of the esophagus (Esophageal Carcinoma, EC) is to seriously endanger the malignant disease of whole mankind's health, global model
Its morbidity and mortality occupies the 8th and the 6th respectively in enclosing.EC mainly has two kinds of histological types：Esophageal squamous cell carcinoma
(Esophageal Squamous Cell Carcinoma, ESCC) and adenocarcinoma of esophagus (Esophageal
Adenocarcinoma, EAC), China's histological type (accounts for more than 90%) based on squamous carcinoma.Announced most according to the World Health Organization
New data shows that ESCC patient newly sends out more than 250,000 people every year in China, and because of about 200,000 people of patient of death from esophageal carcinoma, incidence occupies
All kinds of malignant tumours the 5th in the whole nation, the death rate occupies the 4th, remote super world average level.Although clinically it is used to eat at present
The technological means of the inspection of pipe cancer and treatment is being continuously improved, but the overall 5 years survival rates of patient are still extremely low.Nearly half a century with
Coming, the research on EC pathogenesis both at home and abroad has achieved a series of achievements in mRNA, protein and miRNA fields, but
Its exact pathogenesis is at present still among positive research and probe.(Jemal A, et al.Global cancer
statistics.CA：A cancer journal for clinicians2011,61：69-90；Enzinger PC, et
A1.Esophageal cancer.N Engl J Med2003,349：2241-2252；Old ten thousand green grass or young crops .2004-2005 China dislikes
Property tumor invasion and dead estimation Chinese Journal of Oncology 2009,31：664-668；He Jie, waits esophageal cancer in China epidemiology
Present situation, diagnosis and treatment present situation and future countermeasure Cancer in China magazines, 2011, (7)：501-504.)
After human genome sequencing plan is completed, it was demonstrated that the protein coding gene to receive much concern for a long time only accounts for entirely turning
The 2% of record group, the transcription product more than 98% are ncRNA (noncoding RNA, ncRNA).With RNA groups research into
Exhibition, be initially believed to be subgenomic transcription " noise " ncRNA, progressively confirmed it in several species including humans, very
To be plant physiology and pathologic event in, be respectively provided with extensive and diversified function rarely known by the people.Confirm to have at present to adjust
The ncRNAs of control effect is broadly divided into the microRNAs of the LncRNAs and ＜ 200nt of length ＞ 200nt, although
MicroRNAs is the microRNAs of the LncRNAs and ＜ 200nt for length ＞ 200nt at present, although microRNAs is mesh
Before untill study relatively deep a kind of small ncRNAs, but then it is found that in each species, LncRNAs is not only counted
Amount is big, species is more, and the life such as the transcription and translation in gene, cell differentiation and ontogeny, heredity and epigenetic is lived
Regulating and controlling effect in dynamic is more extensive compared with microRNAs, fine and complicated (Birney 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 surprises from the RNA world.Genes and Development2009,23：1494-
1504；Prasanth KV, et al.Eukaryotic regulatory RNAs：An answer to the′genome
Complexity ' conundrum.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) is more than 200 cores of a kind of transcript length
The non-coding RNA of thuja acid, research in recent years find that it is that one kind has the function of the RNA of important biomolecule, participate in genomic imprinting, dye
A variety of important regulation processes such as colour solid silence, chromatin modification, transcriptional activation, transcription interference, the interior transport of core, in cell differentiation
Important regulating and controlling effect is played with the vital movement such as development, genetic transcription and translation, heredity and epigenetic.In recent years,
More and more authority's researchs confirm that lncRNA plays a part of suppression in the occurrence and development of tumour or promotes tumour, are regulating and controlling
Tumor cell proliferation, apoptosis, cell cycle, invasion and attack transfer ability etc., are respectively provided with particularly significant effect.It is existing at present more
LncRNAs is proved in the mankind including breast cancer, prostate cancer, melanoma, liver cancer, colorectal cancer, carcinoma of urinary bladder etc.
Expression is had differences in kinds of tumors and performs 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
Investigations2013,31：1117-1123；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)by Hepatitis B Virus X Protein Promotes Hepatoma Cell Proliteration 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.).To explore
Whether LncRNA have the function of in ESCC, inventor people ESCC organize and cell line in, with having confirmed at present other
The LncRNA with correlation function carries out pilot study in tumour, it was demonstrated that the LncRNA found in breast cancer tissue
HOTAIR not only ESCC cancers with differential expression in normal tissue, but also it is thin in ESCC cell lines can substantially to suppress tumour
Born of the same parents' apoptosis simultaneously promotes 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, inventor is also confirmed that in prostate
The PlncRNA-1 of middle discovery is significantly raised in ESCC tissues and controllable tumor cell proliferation ability (Wang CM, 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.).
However, tracking document is found in addition to seminar where inventor, the lncRNA on esophageal squamous cell carcinoma is expressed
Functional research of spectrum and its index of correlation is also less at present.For the lncRNA express spectras of system exploration ESCC, find with
The relevant one group of lncRNAs of ESCC occurrence and development specificity, inventor screen one in people's ESCC cancer groups using chip technology
The lncRNA of notable low expression in knitting, the gene are named as RP4-639F20.1 (Ensemble database), its transcriptional domain
Domain is between No. 1 95,426,881-95,428,826bp pairs of chromosome positive-sense strand, full length gene 952bp.Inventor studies
Confirm：Compared with normal tissue, the lncRNA notable low expressions in people's ESCC cancerous tissues, and in the large sample group in later stage
Knit in qRT-PCR (Quantitative Real-time Polymerase Chain Reaction, real-time quantitative PCR) experiments
Expression of the index in people's ESCC cancerous tissues is further confirmed, substantially less than with normal tissue.For ease of the later stage research and
Discuss, inventor is named as HDESCC-lncRNA4 (highly down-regulated in esophageal
Squamous cell carcinoma, long noncoding RNA4).The present inventor has paid close attention to the lncRNA tables of ESCC
Up to spectrum, this kind of new gene regulation factor will be expected to further enrich and improve the tumor invasion including esophageal squamous cell carcinoma
The research of mechanism, also to find that marker, the new cancer target of diagnosing tumor and Index for diagnosis bring hope.
The content of the invention
For the lncRNA express spectras of system research people's esophageal squamous cell carcinoma, find with people's esophageal squamous cell carcinoma occur with
Develop closely related new lncRNAs, by 6 pairs of cancer of the esophagus of inventor's offer and with normal tissue sample, pass through
After RNA extracts kits (RNeasy Micro Kit, article No. 74004) the extraction RNA of Qiagen companies, using Beijing Bo Aosheng
Brilliant core lncRNA chips V2 (4 × 180K) detection of thing Co., Ltd filters out a notable low expression in human esophageal carcinoma
LncRNA, is named as HDESCC-lncRNA4, its gene order is as shown in sequence table SEQ ID NO.1.Later stage is by totally 110 pairs
People ESCC cancers with normal tissue sample qRT-PCR verifications with finding, under the lncRNA is in cancerous tissue in 77 pairs of samples
Mileometer adjustment reaches.The lncRNA is expected to become the marker of diagnosing tumor and Index for diagnosis, while is also provided newly for the treatment of tumour
Target spot.
The object of the present invention is to provide it is a kind of detection in esophageal carcinoma tissue the lncRNA sequences of low expression and
Its application process.
The lncRNA express spectras of the research esophageal squamous cell carcinoma of present system, find to occur with esophageal squamous cell carcinoma
With developing closely related new lncRNA.
The object of the present invention is to provide a kind of HDESCC- for detecting the low expression in esophageal carcinoma tissue
LncRNA4 sequences.
The present invention provides the detection application process of the HDESCC-lncRNA4 sequences, is prepared according to the sequence and is used to eat
The preparation of pipe cancer auxiliary diagnosis or outcome prediction.
3 pairs of primers are used to detect HDESCC-lncRNA4 sequences the present invention according to the HDESCC-lncRNA4 sequence designs
Row.
Pair1 sense primers：SEQ ID NO.2
Pair1 anti-sense primers：SEQ ID NO.3
Pair2 sense primers：SEQ ID NO.4
Pair2 anti-sense primers：SEQ ID NO.5
Pair3 sense primers：SEQ ID NO.6
Pair3 anti-sense primers：SEQ ID NO.7
The present invention is according to the HDESCC-lncRNA4 sequence designs and synthesizes the detection primer for real-time quantitative PCR
Group.The primer sets are suitable for the inspection of SYBR Green, TaqMan probe, molecular beacon, double cross probe, combined probe etc.
Survey.
Being preferably used in the primer sets that dye class real-time quantitative PCR detects is respectively：
Pair1 sense primers：SEQ ID NO.2
Pair1 anti-sense primers：SEQ ID NO.3
In the present invention, qRT-PCR, real-time quantitative PCR, quantitative fluorescent PCR are identical concepts, can be used interchangeably.
It is a further object of the present invention to provide a kind of quantitative fluorescent PCR reagent of detection HDESCC-lncRNA4 expressions
Box and application method, all types fluorescent quantitation gene which is suitable for presently, there are in the market expand
Increase instrument, high sensitivity, it is quantitative quick and precisely, stability it is good, have a good application prospect.
The present invention is prepared for a kind of dye class real-time quantitative PCR kit of detection lncRNA expressions, and component is as follows：
Specific primer, standard DNA template, fluorescent dye, real-time quantitative PCR reaction solution.The wherein described specific primer includes upper
It is SEQ ID NO.2 to swim primer and anti-sense primer, upstream primer sequence, and downstream primer sequence is SEQ ID NO.3.The fluorescence
Quantitative PCR reaction solution includes dNTP, Mg2+, Taq enzyme and buffer buffer solutions.The preferred SYBR Green II of fluorescent dye,
The preferred thermal starting enzyme of Taq enzyme.
It is glimmering the invention also discloses a kind of application method for the dye class PCR kit for fluorescence quantitative for detecting esophageal squamous cell carcinoma
Fluorescent Quantitative PCR system：
Sense primer (10 μm of ol/L) 2 μ L；Anti-sense primer (10 μm of ol/L) 2 μ L；Sample cDNA4 μ L (or standard DNA template
DNA2μL)；50×ROX Reference Dye1μL；2×SYBR Premex Ex Taq II(TIi RNaseH Plus)25μ
L, adds ionized water to 50 μ L.Quantitative fluorescent PCR program：95 DEG C of 30s pre-degenerations, connect 40 circulations：95 DEG C of 5s, 60 DEG C of 30-34s.
The invention also discloses a kind of detection method of long-chain non-coding RNA, include extraction, the sample of sample total serum IgE
The preparation of cDNA, the amplification of HDESCC-lncRNA4.
The extraction of samples described above total serum IgE, the preparation of sample cDNA, the specific steps of HDESCC-lncRNA4 amplifications
Including：
1) extraction of sample total serum IgE：According to Life Technologies companiesReagent (article No.
15596026) reagent needed for and step extraction esophageal carcinoma tissue or the Total RNA of tumour；NanoDrop is used again
ND-1000 nucleic acid quantifications instrument quantitative (NanoDrop Technologies, Wilmington, Delaware) quantifies what is extracted
The purity and concentration of RNA, denaturing formaldehyde gel electrophoresis quality inspection ensure the integrality of the RNA of extraction.
2) preparation of sample cDNA：Using TaKaRa kits PrimeScriptTM RT reagent Kit with
Total serum IgE reverse transcriptions of the gDNA Eraser (Perfect Real Time) (article No. RR047A) to extraction synthesizes cDNA；The reagent
Box includes RNase-Free DNase, can effectively remove the genomic DNA mixed.
The first step removes genomic DNA reaction, and reaction system and condition are as follows：
| Reagent | Usage amount |
| 5×gDNA Eraser Buffer | 2.0μl |
| gDNA Eraser | 1.0μl |
| Total RNA | 1μg |
| RNase Free dH2O | Add water to 10 μ l |
42 DEG C of reaction 2min after above-mentioned component is mixed；
Second step reverse transcription reaction, reaction system and condition are as follows：
| Reagent | Usage amount |
| The reaction solution of step 1 | 10.0μl |
| PrimeScript RT Enzyme Mix I | 1.0μl |
| RT Primer Mix | 1.0μl |
| 5×PrimeScript Buffer 2 | 4μl |
| RNase Free dH2O | 4.0μl |
| Cumulative volume | 20μl |
Above-mentioned component is incubated 15min for 37 DEG C after mixing, then 85 DEG C of inactivation 5sec, that is, obtain cDNA.
3) amplification of HDESCC-lncRNA4：Using TAKARA SYBR Premix Ex TaqTM II(TIi RNaseH
Plus) fluorescence quantitative kit, fluorescent quantitative PCR is carried out by template of the cDNA of reverse transcription.
Dye class quantitative fluorescent PCR system：Sense primer (10 μm of ol/L) 2 μ L；Anti-sense primer (10 μm of ol/L) 2 μ L；Sample
Product cDNA4 μ L (or standard DNA template DNA2 μ L)；50 × ROX Reference Dye (or 50 × ROX Reference Dye
II)1μL；2 × SYBR Premex Ex Taq II (TIi RNaseH Plus) 25 μ L, add ionized water to 50 μ L.Fluorescent quantitation
PCR programs：95 DEG C of 30s pre-degenerations, connect 40 circulations：95 DEG C of 5s, 60 DEG C of 30-34s.
The present invention also have detected kit sensitivity, and the results show kit detection range of the present invention is 107-
102Copies/ μ L, minimum concentrations are 100copies/ μ L.
By the detection to positive, it is 70% to find dye class fluorescence quantitative kit Detection accuracy of the present invention,
Continuous 3 repetitions are tested, and experimental result is stablized.
Brief description of the drawings
Fig. 1 .LncRNA chip dendrograms
6 pairs of esophageal squamous cell carcinoma cancers of display and the LncRNA chips of cancer beside organism's differential expression cluster
Fig. 2 specific primer the selection result figures
Draw for row agarose gel electrophoresis test after 3 pairs of specific primer PCR amplifications of the sequence design of the LncRNA
The effect of thing
The preliminary qRT-PCR testing results (2 of HDESCC-lncRNA4 of first 30 ESCC clinical samples of Fig. 3-ΔctMake
Figure)
The HDESCC-lncRNA4 of Fig. 4 80 ESCC clinical samples of second batch verifies qRT-PCR testing results (2 again-Δct
Mapping)
Totally 110 sample HDESCC-lncRNA4's Fig. 5 two batches unite in cancer and cancer beside organism differential expression qRT-PCR detections
Meter analysis (2-ΔΔctValue compares,*P ＜ 0.05,**P ＜ 0.01)
Embodiment
With reference to specific embodiment, the present invention is further explained, is only used for explaining the present invention, and it is not intended that to this
The limitation of invention.It will be understood by those skilled in the art that：Can in the case where not departing from the principle of the present invention and objective
These embodiments are carried out with a variety of change, modification, replacement and modification, the scope of the present invention is limited by claim and its equivalent
It is fixed.The experimental method of actual conditions is not specified in the following example, usually according to normal condition or according to the bar proposed by manufacturer
Part examinations.
1 people's esophageal squamous cell carcinoma cancer of embodiment and the lncRNA chip expression analysis with normal tissue
One material and method
1st, material
Tissue samples come from inpatient's surgery excision sample of 6 pairs of patients with esophageal squamous cell, and each pair includes food
Pipe tumor tissues and the normal esophageal tissue of pairing.
2nd, method
2.1 tumor tissues and the extraction with normal tissue total serum IgE
Oesophagus is extracted by RNA extracts kits (RNeasy Micro Kit, article No. 74004) specification of Qiagen companies
Phosphorus carninomatosis human tumour tissue and the total serum IgE with normal tissue, the kit include RNase-Free DNase I
(lyophilized), the genomic DNA mixed can effectively be removed.Quantified again with NanoDrop ND-1000 nucleic acid quantification instrument
The purity and concentration of (NanoDrop Technologies, Wilmington, Delaware) quantitative extracted RNA, formaldehyde become
Property gel electrophoresis quality inspection ensure extraction RNA integrality.
2.2 couples of sample RNA carry out fluorescent marker (CRNA expands labelling kit, catalog number：360060-
10)
2.2.1 reverse transcription synthesizes the first chain cDNA
Using Total RNA as starting, T7Oligo (dT) Primer containing T7 promoter sequences is primer, is used
The first chain of CbcScript enzymatic synthesis cDNA.
2.2.2 2 are synthesizednd-strand cDNA
The RNA in heterozygosis chain is cut into short-movie section with RNase H, DNA Polymerase prolong using RNA short-movie sections as primer
Stretch, synthesis 2nd- strand cDNA, and purify double-strand cDNA.
2.2.3 in-vitro transcription synthesizes cRNA
Using cDNA as template, cRNA is synthesized using T7Enzyme Mix；Then purified with RNA Clean-up Kit (MN).
2.2.4 random primed reverse transcription
5ug cRNA are taken, with CbcScript II enzymes, Random Prime carry out reverse transcription, reverse transcription product PCR
NucleoSpinExtractII Kit (MN) are purified.
2.2.5cDNA marked with KLENOW enzymes
Above-mentioned reverse transcription product is taken, KLENOW enzyme marks, marked product PCR are carried out by primer of Random Primer
NucleoSpinExtract II Kit (MN) are purified, and are drained after purification.(Cy5-dCTP or Cy3-dCTP(GE
Healthcare)。
2.3 hybridization and cleaning
The DNA of mark is dissolved in hybridization solution (2 × GEx Hyb Buffer (HI-RPM), 25% formamide), miscellaneous in 45 DEG C
Hand over overnight.After hybridization, first contain 0.2%SDS at 42 DEG C or so, 5min is washed in the liquid of 2 × SSC, then in 0.2 × SSC
Middle room temperature washes 5min.It can be used to scan after slide drying.
2.4 chip scanning
Chip is scanned with Agilent G2565CA Microarray Scanner, obtains hybridization picture.
The collection and data analysis of 2.5 chip images
2.5.1 fluorescence signal value is extracted
Chip image is analyzed using Feature Extraction image analysis softwares, picture signal is converted into
Digital signal.
2.5.2 differential gene screens
Initial data is input in GeneSpring GX softwares, using percentile shift methods to signal value
It is normalized.Then Absolute Fold change >=2 are used, while Flag is carried out labeled as the standard of Detected
Differential gene screens.
Two results
Fig. 1 is shown in LncRNA chip cluster analyses on people's esophageal squamous cell carcinoma.Chip examination is found in a plurality of expression
The lncRNAs that reconciliation expression is lowered.Wherein HDESCC-lncRNA4 shows to express in cancerous tissue and significantly lowers, in view of it can
Can be in the cancerous tissue of people's esophageal squamous cell carcinoma there are specific expressed, the present invention uses extensive sample by following embodiments
This carries out the repeated authentication of index in batches.
Embodiment 2qRT-PCR preliminary identifications HDESCC-lncRNA4 is in the cancerous tissue of esophageal squamous cell carcinoma and normal group
Differential expression in knitting
First, experiment material
Other 30 are chosen again to the cancerous tissue of the sample of chip testing (be different from) people's esophageal squamous cell carcinoma and with aligning
Often tissue, qRT-PCR preliminary identifications are carried out to the differential expression of HDESCC-lncRNA4.
2nd, experimental method and result
1 primer specificity is identified
The screening of 1.1 specific primers
(1) the relevant transcript sequences of HDESCC-lncRNA4 are extracted from Ensemble databases, and with according to transcript
Sequence primer is designed by the design of primers instrument (Primer-BLAST) of NCBI；
(2) primer after designing is evaluated with Oligo7,3 pairs of primers of every design；
Pair1 sense primers：SEQ ID NO.2
Pair1 anti-sense primers：SEQ ID NO.3
Pair2 sense primers：SEQ ID NO.4
Pair2 anti-sense primers：SEQ ID NO.5
Pair3 sense primers：SEQ ID NO.6
Pair3 anti-sense primers：SEQ ID NO.7
Being preferably used in the primer sets that dye class real-time quantitative PCR detects is respectively：
Pair1 sense primers：SEQ ID NO.2
Pair1 anti-sense primers：SEQ ID NO.3
(3) by the cancer of people's esophageal squamous cell carcinoma with matching somebody with somebody normal tissue according to Life Technologies companiesReagent needed for reagent (article No. 15596026) and step extraction total serum IgE；NanoDrop ND-1000 nucleic acid is used again
The purity of quantitative the extracted RNA of quantitative instrument quantitative (NanoDrop Technologies, Wilmington, Delaware) and
Concentration, denaturing formaldehyde gel electrophoresis quality inspection ensure the integrality of the RNA of extraction.
(4) TaKaRa kits PrimeScript is usedTM RT reagent Kit with gDNA Eraser
The total serum IgE reverse transcription of (Perfect Real Time) (article No. RR047A) to extraction synthesizes cDNA, which includes
GDNAEraser DNase, can effectively remove the genomic DNA mixed.
The first step removes genomic DNA reaction, and reaction system and condition are as follows：
| Reagent | Usage amount |
| 5×gDNA Eraser Buffer | 2.0μl |
| gDNA Eraser | 1.0μl |
| Total RNA | 1μg |
| RNase Free dH2O | Add water to 10 μ l |
42 DEG C of reaction 2min after above-mentioned component is mixed；
Second step reverse transcription reaction, reaction system and condition are as follows：
| Reagent | Usage amount |
| The reaction solution of step 1 | 10.0μl |
| PrimeScript RT Enzyme Mix I | 1.0μl |
| RT Primer Mix | 1.0μl |
| 5×PrimeScript Buffer 2 | 4μl |
| RNase Free dH2O | 4.0μl |
| Cumulative volume | 20μl |
Above-mentioned component is incubated 15min for 37 DEG C after mixing, then 85 DEG C of inactivation 5sec, that is, obtain cDNA.With synthesis
CDNA be template, for 3 pairs of primers setting reaction groups and the negative control groups of cDNA templates is not added with respectively, with except outer primer
Dimer is possible, then carries out PCR reactions by the primer of step (2) design；
(5) electrophoresis detection, selects Marker DL1000 (TaKaRa).Primer selection standard：A. by Marker to amplification
Clip size carries out entry evaluation, and amplified fragments size is identical with expection；B. amplified production only has one, that is, ensures primer amplification
Specificity.The results are shown in Figure 2, and optimal primer pair is Pair1, the specific primer of upstream, its sequence is shown in sequence table SEQ
ID NO.2, the specific primer in downstream, its sequence are shown in sequence table SEQ ID NO.3.
The extraction of 2 sample total serum IgEs：
Using liquid nitrogen grinding method, according to Life Technologies companiesReagent (article No.
15596026) reagent needed for and step extraction esophageal carcinoma tissue or the Total RNA of tumour.Main operational steps are such as
Under：
(1) in liquid nitrogen, when extracting RNA is put into tissue in the mortar of precooling to carry out quick freeze sample after in vitro
Grinding, the liquid feeding nitrogen in grinding, whole process all not make liquid nitrogen volatilization dry；
(2) after tissue sample is ground into powder, when liquid nitrogen is evaporated completely substantially, 2- is added in each mortar
3mlTRIZOL reagents, TRIZOL can be frozen into solid-like after adding, can continue to grind this solid into powder, with mortar temperature
Degree goes back up to room temperature, and the TRIZOL of solid-like is progressively returned to liquid condition, this TRIZOL agent transfer to glass homogenizer
In, further it is homogenized 3-5min；
(3) TRIZOL agent transfers into 1.5ml centrifuge tubes, every 1 milliliter of TRIZOL reagents add 0.2 milliliter of chlorine
It is imitative.Carefully cover sample cell.Shake pipe energetically with hand 15 seconds, 15-30 DEG C is incubated 2-3 minutes.2-8 DEG C, centrifuged no more than 12000g
15 minutes.After centrifugation, mixture is separated into the colourless aqueous phase on red lower floor (phenol-chloroform phase), interphase and upper strata, and RNA is deposited
It is water phase；
(4) water is mutually transferred to a new pipe, and for mixing isopropanol to precipitate RNA from water phase, every 1 milliliter is used for initially same
The TRIZOL reagents of matter use 0.5 milliliter of isopropanol, 15-30 DEG C of samples of incubation 10 minutes, 2-8 DEG C, no more than 12000g from
The heart 10 minutes；
(5) supernatant is abandoned, RNA precipitate is washed once with 75% ethanol, every milliliter of TRIZOL examination for being used for initial homogeneity
Using at least 1 milliliter 75% of ethanol, vortex mixed, centrifuges 5 minutes no more than 7500g by 2-8 DEG C for agent；
(6) supernatant is abandoned, simply dry RNA precipitate (be air-dried or be dried in vacuo 5-10 minutes), with 20-100ul without RNA
The water piping and druming of enzyme dissolves RNA and is stored in -70 DEG C several times, in this process it may be noted that not allow RNA precipitate to be completely dried, because
Its solubility will be substantially reduced for this.
The preparation of 3 standard DNA templates
To specifications, matching somebody with somebody in normal tissue from esophageal squamous cell carcinoma, Invitrogen RTIZOL reagents are utilized
Box extracts total serum IgE, and further usesRNA clean-up kits (MACHEREY-NAGEL,
Germany column purification) was carried out to total serum IgE, then carries out reverse transcription reaction, reverse transcription system is：Reverse transcription random primer (2 μ
Mol/L) 1 μ L, 4 μ L, dNTP mixture (every kind of 2.5mmol/L) of total serum IgE 4 μ L, 0.1mol/L DTT2 μ L, SuperScript
2 μ L (TAKARA) reaction condition of RNase H reverse transcriptases (200U/ μ L) is：37 DEG C of water-baths 60min, 95 DEG C of 3min.
The cDNA that reverse transcription reaction is obtained carries out Standard PCR, and reaction system and condition are as follows：10×Ex Taq
Buffer10 μ L, dNTP Mixture (each 2.5mmol/L) 4 μ L, Sequence NO.2 (1O pmol) 4 μ L, Sequence
0.5 μ L of NO.3 (10pmol) 4 μ L, cDNA (0.1-2 μ L) 5 μ L, Ex Taq archaeal dna polymerases, distilled water polishing to 100uL.Reaction
Condition is 94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 50s, 50 DEG C of annealing 50s, 72 DEG C of extensions 50s, 35cycles；Last 72 DEG C are prolonged
Stretch 10min.
5 μ L are sampled, the product of PCR amplification is detected into row agarose gel electrophoresis, gel extraction is carried out and purifies (recycling
Use kit：EZ-10 Spin Column DNA Gel Extraction Kit), purified product is connected to pGM-T grams
Grand carrier, is then transformed into DH5 α competent cells.Pass through the specificity that sequence is SEQ ID NO.2 and SEQ ID NO.3
Primer screening positive colony.Plasmid DNA is extracted after positive colony amplification, Plasmid DNA is determined using NanoDrop ND-1000 nucleic acid
Amount instrument quantifies (NanoDrop Technologies, Wilmington, Delaware) and does 10 times and is serially diluted as standard items
For the preparation of standard curve, (standard DNA template concentration range is 108-102copies/μl)。
4 sensitivity experiments
Recombinant plasmid is taken to be diluted to 10 in proportion8、107、106、105、104、103、102A copy/μ L, carries out fluorescent quantitation
PCR, the detection sensitivity using the least concentration of test positive as this method.This research institute establish method detection range be
108-102Copies/ μ L, minimum concentrations are 100copies/ μ L.
5 synthesis cDNA templates
Above-mentioned 30 pairs of esophageal squamous cell carcinoma cancerous tissues and the total serum IgE with normal tissue are taken, using TaKaRa kits
PrimeScriptTMRT reagent Kit with gDNA Eraser (Perfect Real Time) (article No. RR047A) are right
The total serum IgE reverse transcription synthesis cDNA of extraction, the kit include gDNA Eraser DNase, can effectively remove the gene mixed
Group DNA.
The first step removes genomic DNA reaction, and reaction system and condition are as follows：
| Reagent | Usage amount |
| 5×gDNA Eraser Buffer | 2.0μl |
| gDNA Eraser | 1.0μl |
| Total RNA | 1μg |
| RNase Free dH2O | Add water to 10 μ l |
42 DEG C of reaction 2min after above-mentioned component is mixed；
Second step reverse transcription reaction, reaction system and condition are as follows：
| Reagent | Usage amount |
| The reaction solution of step 1 | 10.0μl |
| PrimeScript RT Enzyme Mix I | 1.0μl |
| RT Primer Mix | 1.0μl |
| 5×PrimeScript Buffer 2 | 4μl |
| RNase Free dH2O | 4.0μl |
| Cumulative volume | 20μl |
Above-mentioned component is incubated 15min for 37 DEG C after mixing, then 85 DEG C of inactivation 5sec, that is, obtain cDNA.
6 dye class fluorescence quantitative PCR detection HDESCC-lncRNA4 expression quantity
Using TAKARA SYBR Premix Ex TaqTMII (TIi RNaseH Plus) fluorescence quantitative kit (article No.
RR820A).50 μ L qRT-PCR reaction systems include：Sense primer (10 μm of ol/L) 2 μ L；Anti-sense primer (10 μm of ol/L) 2 μ L；
4 μ L of sample cDNA；50×ROX Reference Dye 1μL；2×SYBR Premex Ex Taq II(TIi RNaseH
Plus) 25 μ L, add ionized water to 50 μ L.Instrument uses Applied Biosystems 7500.Quantitative fluorescent PCR program：95℃
30s pre-degenerations, connect 40 circulations：95 DEG C of 5s, 60 DEG C of 30-34s.
According to the relative quantification formula of qRT-PCR：2-ΔCt, compare HDESCC-lncRNA4 in patients with esophageal squamous cell carcinoma tumor group
Knit and with the expression in normal tissue.The results are shown in Figure 3：QRT-PCR stable amplification results, wherein HDESCC-
The expressions of lncRNA4 in the normal tissue are between 0.002-0.04, hence it is evident that higher than tumor tissue and control plasmid
100copies, and the expression quantity of the HDESCC-lncRNA4 in tumor tissue these results suggest that between 0.000-0.003
HDESCC-lncRNA4 common lower expressions in tumor tissues.It is defined as according still further to relative expression quantity T-N ＞ 0 in index expression
Adjust；T-N ＜ 0 are defined as index expression and lower.Then this experimental result is shown：HDESCC-lncRNA4 is in 30 pairs of ESCC cancer groups
In knitting, 21 pairs of the expression downward compared with normal tissue, according still further to formula：Lower expression number of cases/total detection number of cases x100%
The positive rate of the index is defined, then the positive rate of the index is 70%.
Embodiment 3qRT-PCR further verifies HDESCC-lncRNA4 in the cancerous tissue of esophageal squamous cell carcinoma and normal
Differential expression in tissue
1st, qRT-PCR kit forms
1.1 dye class HDESCC-lncRNA4qRT-PCR kit forms：
(1) sense primer：SEQ ID NO.2
(2) anti-sense primer：SEQ ID NO.3；
Other reagents are with reference to SYBR Premix Ex TaqTMII (Tli RNaseH Plus) fluorescence quantitative kit
(Code No.RR820A)。
The detection of 2.HDESCC-lncRNA4qRT-PCR
The preparation of 2.1 total serum IgEs
The cancerous tissue of other 80 couples of esophageal squamous cell carcinoma patients is chosen and with normal tissue, according to Life
Technologies companiesReagent needed for reagent (article No. 15596026) and step extraction total serum IgE, specific ginseng
See specification.Again with NanoDrop ND-1000 nucleic acid quantifications instrument it is quantitative (NanoDrop Technologies, Wilmington,
Delaware) purity and concentration of quantitative extracted RNA, denaturing formaldehyde gel electrophoresis quality inspection ensure the integrality of the RNA of extraction.
2.2cDNA synthesis
Using TaKaRa kits PrimeScriptTM RT reagent Kit with gDNA Eraser(Perfect
Real Time) (article No. RR047A), qualified total serum IgE will be detected and carry out reverse transcription reaction.
2.3qRT-PCR detection
QRT-PCR uses Applied Biosystems7500 in instrument.It is glimmering in qRT-PCR response procedures such as embodiment 2
Photoinitiator dye class real-time quantitative PCR detects HDESCC-lncRNA4 expression quantity.
3 testing results
Real-time quantitative PCR testing result is shown, with matching normal group in the sample of 80 couples of esophageal squamous cell carcinoma patients
Knit and compare, 56 pairs of patient indexs express obvious downward in tumor tissues, and Positive rate is 70% (Fig. 4).We are to upper
State sample to carry out being repeated 3 times qRT-PCR inspections, as a result repeatability shows the repeated of kit of the present invention and stablize up to 100%
Property is preferable.
Embodiment 4HDESCC-lncRNA4 is analyzed in the potential value of ESCC diagnosis and Index for diagnosis
HDESCC-lncRNA4 have detected on the basis of the expression of 110 ESCC patients, qRT-PCR is being reacted
As a result SPSS For Windows20.0 softwares are used, related data is examined by Normal test, is selected according to data type
Mann-Whitney, which is examined, carries out data analysis, and P ＜ 0.05 think that the differential expression of the index is statistically significant.QRT-PCR
Expressions of the interpretation of result HDESCC-lncRNA4 in 110 pairs of ESCC samples, which is substantially less than, matches normal esophageal tissue (figure
5, P ＜ 1 × 10-6).With going deep into for later stage result of study, functions and its mechanism of action of the HDESCC-lncRNA4 in ESCC
To progressively it illustrate, this novel long-chain non-coding RNA, which can not only become, diagnoses relevant biomarker, is more expected to become new
ESCC therapy targets to improve, improve clinical ESCC therapeutic effects.Obviously, find more, more accurately as HDESCC-
It is expected to participate in auxiliary ESCC diagnosis, treatment and the relevant biomarker of prognosis as lncRNA4, has highly important existing
Sincere justice.
Claims (5)
1. a kind of detection reagent of long-chain non-coding RNA is being prepared in esophageal squamous cell carcinoma auxiliary diagnosis or curative effect predication reagent
Application, the nucleotides sequence of the long-chain non-coding RNA is classified as SEQ ID NO.1.
2. application according to claim 1, it is characterised in that described is pre- for esophageal squamous cell carcinoma auxiliary diagnosis or curative effect
The reagent of survey is real-time quantitative PCR detection reagent.
A kind of 3. real time quantitative PCR detecting reagent kit for being used for esophageal squamous cell carcinoma auxiliary diagnosis or outcome prediction, it is characterised in that
Long-chain non-coding RNA including being classified as SEQ ID NO.1 according to nucleotides sequence, which designs and synthesizes out, is specifically used for real-time quantitative
The detection primer of PCR；Specific primer is as shown in SEQ ID NO.2 in sequence table and SEQ ID NO.3.
4. applying according to claim 1, the detection method of long-chain non-coding RNA includes the extraction of sample RNA, sample cDNA
Preparation, the amplification of lncRNA.
5. application according to claim 4, it is characterised in that the extraction of the sample RNA, the preparation of sample cDNA,
The specific steps of the amplification of lncRNA include：
1) extraction of sample total serum IgE：According to Life Technologies companiesReagent needed for reagent and step carry
Take esophageal carcinoma tissue or the Total RNA of tumour；Carried again with NanoDrop ND-1000 nucleic acid quantifications instrument is quantitative
The purity and concentration of the RNA taken, denaturing formaldehyde gel electrophoresis quality inspection ensure the integrality of the RNA of extraction；
2) preparation of sample cDNA：Using TaKaRa kit PrimeScriptTM RT reagent Kit with gDNA
Total serum IgE reverse transcriptions of the Eraser to extraction synthesizes cDNA；The kit includes RNase-Free DNase, can effectively remove and mix
Genomic DNA；
3) amplification of lncRNA：Using TAKARA SYBR Premix Ex TaqTMII fluorescence quantitative kits, with reverse transcription
CDNA carries out fluorescent quantitative PCR for template.
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| CN104357443B (en) * | 2014-11-25 | 2017-03-15 | 中国科学院生物物理研究所 | A kind of detection of the long-chain non-coding RNA for bladder cancer examination and its application |
| CN104845968A (en) * | 2015-03-27 | 2015-08-19 | 武汉华美生物工程有限公司 | Long-chain non-coding RNA cloning method |
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