CN103952480B - Method for detecting LIFR-AS1 of esophageal squamous cell carcinoma patient, and its application - Google Patents
Method for detecting LIFR-AS1 of esophageal squamous cell carcinoma patient, and its application Download PDFInfo
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Abstract
The invention relates to a long noncoding RNA and its application. A specific real-time quantitative PCR primer and a specific real-time quantitative probe are designed and synthesized according to the long noncoding RNA sequence in order to prepare a preparation used for the auxiliary diagnosis or the curative effect prediction of esophageal cancer. A result of the detection of the expression level of the long noncoding RNA in a clinic medical case sample of the esophageal cancer by utilizing the real-time quantitative PCR preparation shows that the expression level of the long noncoding RNA in the esophageal cancer substantially decreases. The method is expected to prepare preparations used for the auxiliary diagnosis and the curative effect predication or the prognosis determination of the esophageal cancer.
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 concrete and
Speech, the present invention relates to a kind of long-chain non-coding RNA is preparing cancer of the esophagus auxiliary diagnosis or the application in prognosis preparation.
Background technology
The cancer of the esophagus (Esophageal Carcinoma, EC) be the serious harm whole mankind health malignant disease, global model
Its M ＆ M occupies respectively the 8th and the 6th 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.Announce most according to the World Health Organization
New data shows that ESCC patient newly sends out every year more than 250,000 people in China, and because of the people of patient about 200,000 of death from esophageal carcinoma, the incidence of disease is occupied
All kinds of malignant tumours the 5th in the whole nation, the death rate occupies the 4th, far super world average level.Although being clinically used to eat at present
The technological means of the inspection of pipe cancer and treatment is updating, but 5 years survival rates of overall patient are still extremely low.Nearly half a century with
Come, the research with regard to EC pathogenesis both at home and abroad achieves a series of achievements in mRNA, protein and miRNA fields, 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 year China dislike
Property tumor invasion with death 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 magazine, 2011, (7)：501-504.)
After human genome sequencing plan is completed, it was demonstrated that the protein coding gene for receiving much concern for a long time only accounts for whole turning
The 2% of record group, the transcription product more than 98% is ncRNA (noncoding RNA, ncRNA).With entering that RNA groups are studied
Exhibition, is initially believed to the ncRNA for being subgenomic transcription " noise ", is 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 at present to have to adjust
The ncRNAs of control effect is broadly divided into length>The LncRNAs of 200nt and<The microRNAs of 200nt, although microRNAs
It is to study the relatively deep little ncRNAs of a class so far, but subsequently it is found that in each species, LncRNAs is not
Only quantity is big, species is more, and the transcription and translation in gene, cell differentiation and ontogeny, heredity and epigenetic etc. are raw
Regulating and controlling effect in life activity is more extensive compared with microRNAs, fine and complexity (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 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 a class transcript length more than 200 cores
The non-coding RNA of thuja acid, research in recent years finds that it is the RNA that a class has important biomolecule function, participates in genomic imprinting, dye
Various 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, hereditary and epigenetic.In recent years,
Increasing authority's research confirms that lncRNA plays a part of to suppress or promote tumour in tumour develops, in regulation and control
The aspects such as tumor cell proliferation, apoptosis, cell cycle, invasion and attack transfer ability, are respectively provided with particularly significant effect.It is existing at present more
LncRNAs is proved the mankind including including breast cancer, prostate cancer, melanoma, liver cancer, colorectal cancer, carcinoma of urinary bladder etc.
Have differences in kinds of tumors and express and perform 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 has function in ESCC, inventor uses current in people ESCC tissues and clone
Have confirmed that the LncRNA with correlation function carries out pilot study in other tumours, it was demonstrated that find in breast cancer tissue
LncRNA HOTAIR not only ESCC cancers with differential expression in normal tissue, and can be obvious in ESCC cell lines
Suppress apoptosis of tumor cells and 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.).Additionally, inventor also confirms that
The PlncRNA-1 found in prostate is significantly raised and controllable tumor cell proliferation ability (Wang in ESCC tissues
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, follow the trail of document to find in addition to the seminar of inventor place, to express with regard to the lncRNA of esophageal squamous cell carcinoma
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
There is one group of related lncRNAs of development specificity in ESCC, inventor screens one in people's ESCC cancer groups using chip technology
The lncRNA of notable low expression in knitting, the gene is named as LIFR-ASl (Ensemble database), its transcript regions position
Between No. 5 chromosome positive-sense strands 38,559,044-38,579,696bp pairs, full length gene is 685bp.Inventor's research card
It is real：Compared with normal tissue, the lncRNA notable low expressions in people's ESCC cancerous tissues, and in the large sample tissue in later stage
Enter in qRT-PCR (Quantitative Real-time Polymerase Chain Reaction, real-time quantitative PCR) experiments
One step confirms expression of the index in people's ESCC cancerous tissues, substantially less than with normal tissue.Study for ease of the later stage and discuss
State, inventor is named as HDESCC-lncRNA7 (highly down-regulated in esophageal
squamous cell carcinoma,long noncoding RNA7).The present inventor has paid close attention to the lncRNA tables of ESCC
Up to spectrum, the new gene regulation factor of this class will be expected to further enrich and improve including the tumor invasion including esophageal squamous cell carcinoma
The research of mechanism, also brings hope to find the mark of diagnosing tumor and Index for diagnosis, new cancer target.
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
The closely related new lncRNAs of development, by 6 pairs of cancer of the esophagus of inventor's offer and with normal tissue sample, passes through
The RNA extracts kits (RNeasy Micro Kit, article No. 74004) of Qiagen companies are extracted after RNA, using Beijing Bo Aosheng
Brilliant core lncRNA chip V2 (4 × 180K) detection of thing Co., Ltd filters out a notable low expression in human esophageal carcinoma
LncRNA, is named as HDESCC-lncRNA7, and its gene order is as shown in sequence table SEQ ID NO.1.Later stage passes through common 11O pair
People ESCC cancers find that the lncRNA is expressed in cancerous tissue in 87 pairs of samples with normal tissue sample qRT-PCR checkings
Significantly lower.The lncRNA is expected to become the mark of diagnosing tumor and Index for diagnosis, while also the treatment for tumour is provided
New target spot.
It is an object of the invention 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 occur with esophageal squamous cell carcinoma
The new lncRNA closely related with development.
It is an object of the invention to provide a kind of HDESCC- of detection low expression in esophageal carcinoma tissue
LncRNA7 sequences.
The invention provides the detection application process of the HDESCC-lncRNA7 sequences, prepares for eating according to the sequence
The preparation of pipe cancer auxiliary diagnosis or outcome prediction.
3 pairs of primers are used to detect HDESCC-lncRNA7 sequences the present invention according to the HDESCC-lncRNA7 sequences Designs
Row.
Pairl upstream primers：SEQ ID NO.2
Pairl downstream primers：SEQ ID NO.3
Pair2 upstream primers：SEQ ID NO.4
Pair2 downstream primers：SEQ ID NO.5
Pair3 upstream primers：SEQ ID NO.6
Pair3 downstream primers：SEQ ID NO.7
The present invention is according to the HDESCC-lncRNA7 sequences Designs and synthesizes detection primer for real-time quantitative PCR
Group.The primer sets are applied to the inspection of SYBR Green, TaqMan probe, molecular beacon, double cross probe, combined probe etc.
Survey.
The primer sets for being preferably used in the detection of dye class real-time quantitative PCR are respectively：
Pair2 upstream primers：SEQ ID NO.4
Pair2 downstream primers：SEQ ID NO.5
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-lncRNA7 expressions
Box and using method, all types fluorescent quantitation gene that the PCR kit for fluorescence quantitative is suitable for presently, there are on market expands
Increase instrument, sensitivity is high, quantitatively quick and precisely, good stability, 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 reactant liquor.Wherein described specific primer includes upper
Trip primer and downstream primer, upstream primer sequence is SEQ ID NO.4, and downstream primer sequence is SEQ ID NO.5.The fluorescence
Quantitative PCR reactant liquor includes dNTP, Mg2+, Taq enzyme and buffer buffer solutions.The preferred SYBR Green II of the fluorescent dye,
The preferred thermal starting enzyme of Taq enzyme.
The invention also discloses a kind of using method of the dye class PCR kit for fluorescence quantitative of detection esophageal squamous cell carcinoma, glimmering
Fluorescent Quantitative PCR system：
Upstream primer (10 μm of ol/L) 2 μ L；Downstream 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, plus ionized water is to 50 μ L.Quantitative fluorescent PCR program：95 DEG C of 30s denaturations, 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, including extraction, the sample of sample total serum IgE
The preparation of cDNA, the amplification of HDESCC-lncRNA7.
The extraction of samples described above total serum IgE, the preparation of sample cDNA, the concrete steps of HDESCC-lncRNA7 amplifications
Including：
1) extraction of sample total serum IgE：According to Life Technologies companiesReagent (article No.
15596026) reagent needed for and step extract the Total RNA of esophageal carcinoma tissue or tumour；NanoDrop is used again
ND-1000 nucleic acid quantifications instrument quantitative (NanoDrop Technologies, Wilmington, Delaware) is quantitatively extracted
The purity and concentration of RNA, denaturing formaldehyde gel electrophoresis quality inspection guarantees the integrality of the RNA for extracting.
2) preparation of sample cDNA：Using TaKaRa kit PrimeScriptTM RT reagent Kit with
GDNA Eraser (Perfect Real Time) (article No. RR047A) synthesize cDNA to the total serum IgE reverse transcription extracted；The reagent
Box includes RNase-Free DNase, can effectively remove the genomic DNA for mixing.
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 it is as follows：
| Reagent | Usage amount |
| The reactant liquor of step 1 | 10.0μl |
| PrimeScript RT Enzyme Mix I | 1.0ul |
| RT Primer Mix | 1.0μl |
| 5×PrimeScript Buffer2 | 4μl |
| RNase Free dH2O | 4.0ul |
| Cumulative volume | 20μl |
37 DEG C of incubation 15min after above-mentioned component is well mixed, then 85 DEG C of inactivation 5sec, that is, obtain cDNA.
3) amplification of HDESCC-lncRNA7：Using TAKARA SYBR Premix Ex TaqTM II(TIi RNaseH
Plus) fluorescence quantitative kit, by template of the cDNA of reverse transcription fluorescent quantitative PCR is carried out.
Dye class quantitative fluorescent PCR system：Upstream primer (10 μm of ol/L) 2 μ L；Downstream 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, plus ionized water is to 50 μ L.Fluorescent quantitation
PCR programs：95 DEG C of 30s denaturations, connect 40 circulations：95 DEG C of 5s, 60 DEG C of 30-34s.
The present invention also have detected kit sensitivity, as a result show that 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 found that dye class fluorescence quantitative kit Detection accuracy of the present invention is 73%-
81%, continuous 3 repetitions are tested, and experimental result is stable.
Description of the drawings
Fig. 1 .LncRNA chip dendrograms
Show that 6 pairs of esophageal squamous cell carcinoma cancers are clustered with the LncRNA chips of cancer beside organism's differential expression
Fig. 2. specific primer the selection result figure
Draw for row agarose gel electrophoresis test after 3 couples of specific primer PCR amplifications of the sequences Design of the LncRNA
The effect of thing
Fig. 3. the preliminary qRT-PCR testing results (2 of HDESCC-lncRNA7 of first 30 ESCC clinical samples-ΔctMake
Figure)
Fig. 4. the HDESCC-lncRNA7 of 80 ESCC clinical samples of second batch verifies again qRT-PCR testing results (2-Δct
Mapping)
Fig. 5. totally 110 sample HDESCC-lncRNA7's two batches unite in cancer and cancer beside organism's differential expression qRT-PCR detections
Meter analysis (2-ΔΔctValue compares,*P<0.05,**P<0.01)
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further, is only used for explaining the present invention, and it is not intended that to this
The restriction of invention.It will be understood by those skilled in the art that：Can in the case of the principle and objective without departing from the present invention
So that these embodiments are carried out with various changes, modification, replacement and modification, the scope of the present invention is limited by claim and its equivalent
It is fixed.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition or according to the bar proposed by manufacturer
Part examinations.
The people's esophageal squamous cell carcinoma cancer of embodiment 1 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 of pairing tissue.
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, the article No. 74004) specification of Qiagen companies
Phosphorus carninomatosis human tumour is organized and the total serum IgE with normal tissue, and the kit includes RNase-Free DNase I
(lyophilized), the genomic DNA for mixing can effectively be removed.It is quantitative with NanoDrop ND-1000 nucleic acid quantification instrument again
The purity and concentration of the RNA that (NanoDrop Technologies, Wilmington, Delaware) is quantitatively extracted, formaldehyde becomes
Property gel electrophoresis quality inspection guarantee extract RNA integrality.
The 2.2 couples of sample RNA carry out fluorescence labeling (CRNA amplification label kits, catalog number：360060-
10)
2.2.1 reverse transcription synthesizes the first chain cDNA
With Total RNA as starting, the T7Oligo containing T7 promoter sequences (dT) Primer is primer, is used
CbcScript enzymatic synthesis the first chain cDNA.
2.2.2 2 are synthesizednd-strand cDNA
The RNA in heterozygosis chain is cut into into short-movie section with RNase H, DNA Polymerase are prolonged with RNA short-movie sections as primer
Stretch, synthesis 2nd- strand cDNA, and purify double-strand cDNA.
2.2.3 in-vitro transcription synthesizes cRNA
With cDNA as template, using T7Enzyme Mix cRNA is synthesized；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
NucleoSpinExtract II Kit (MN) is purified.
2.2.5cDNA marked with KLENOW enzymes
Above-mentioned reverse transcription product is taken, by primer of Random Primer KLENOW enzyme marks, marked product PCR are carried out
NucleoSpinExtract II Kit (MN) is purified, and is 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 terminates, 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.Slide can be used to scan after drying.
2.4 chip scanning
Chip is scanned with Agilent G2565CA Microarray Scanner, obtains hybridizing 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
Data signal.
2.5.2 differential gene screening
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 the standard that Flag is labeled as Detected is carried out
Differential gene is screened.
Two results
Fig. 1 is shown in LncRNA chip cluster analyses with regard to people's esophageal squamous cell carcinoma.Chip examination is found in a plurality of expression
The lncRNAs that the expression that reconciles is lowered.Wherein HDESCC-lncRNA7 shows to be expressed in cancerous tissue and significantly lowers, in view of it can
Can exist in the cancerous tissue of people's esophageal squamous cell carcinoma it is specific expressed, the present invention by following examples adopt extensive sample
This carries out in batches the repeated authentication of index.
Cancerous tissues and normal group of the embodiment 2qRT-PCR preliminary identification HDESCC-lncRNA7 in esophageal squamous cell carcinoma
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, to the differential expression of HDESCC-lncRNA7 qRT-PCR preliminary identifications are carried out.
2nd, experimental technique and result
1 primer specificity is identified
1.1 the screening of specific primer
(1) the related transcript sequences of HDESCC-lncRNA7 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, and 3 pairs of primers are designed per bar；
Pair1 upstream primers：SEQ ID NO.2
Pair1 downstream primers：SEQ ID NO.3
Pair2 upstream primers：SEQ ID NO.4
Pair2 downstream primers：SEQ ID NO.5
Pair3 upstream primers：SEQ ID NO.6
Pair3 downstream primers：SEQ ID NO.7
Jing RT-PCR and agarose gel electrophoresis experimental verification, screen the primer for the detection of dye class real-time quantitative PCR
Group is following (Fig. 2)：
Pair2 upstream primers：SEQ ID NO.4
Pair2 downstream primers：SEQ ID NO.5
(3) by the cancer of people's esophageal squamous cell carcinoma with normal tissue according to Life Technologies companiesReagent needed for reagent (article No. 15596026) and step extract total serum IgE；NanoDrop ND-1000 nucleic acid is used again
The purity of the RNA that quantitative instrument quantitative (NanoDrop Technologies, Wilmington, Delaware) is quantitatively extracted and
Concentration, denaturing formaldehyde gel electrophoresis quality inspection guarantees the integrality of the RNA for extracting.
(4) using TaKaRa kit PrimeScriptTM RT reagent Kit with gDNA Eraser
(Perfect Real Time) (article No. RR047A) synthesizes cDNA to the total serum IgE reverse transcription extracted, and the kit includes gDNA
Eraser DNase, can effectively remove the genomic DNA for mixing.
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 it is as follows：
| Reagent | Usage amount |
| The reactant liquor of step 1 | 10.0μl |
| PrimeScript RT Enzyme Mix I | 1.0μl |
| RT Primer Mix | 1.0μl |
| 5×PrimeScript Buffer2 | 4μl |
| RNase Free dH2O | 4.0μl |
| Cumulative volume | 20μl |
37 DEG C of incubation 15min after above-mentioned component is well mixed, then 85 DEG C of inactivation 5sec, that is, obtain cDNA.To synthesize
CDNA be template, be respectively directed to 3 pairs of primers setting reaction groups and be not added with the negative control group of cDNA templates, with except outer primer
Dimer is possible, then the primer designed by step (2) enters performing PCR reaction；
(5) electrophoresis detection, from 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 one of which, that is, ensure primer amplification
Specificity.As a result as shown in Fig. 2 optimal primer pair is Pair2, the specific primer of upstream, its sequence is shown in sequence table SEQ
ID NO.4, the specific primer in downstream, its sequence is shown in sequence table SEQ ID NO.5.
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 extract the Total RNA of esophageal carcinoma tissue or tumour.Main operational steps are such as
Under：
(1) in vitro rear quick freeze in liquid nitrogen, tissue is put in the mortar of precooling during extracting RNA carried out sample
Grinding, adds liquid nitrogen, whole process all to make liquid nitrogen volatilization dry in grinding；
(2) after tissue sample is ground into powder, when liquid nitrogen is evaporated completely substantially, in each mortar 2- is added
3mlTRIZOL reagents, TRIZOL can be frozen into solid-like after adding, and 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) in TRIZOL agent transfers to 1.5ml centrifuge tubes, the TRIZOL reagents per 1 milliliter add 0.2 milliliter of chlorine
It is imitative.Carefully cover sample cell.Pipe is shaken energetically 15 seconds, 15-30 DEG C of incubation 2-3 minute with hand.2-8 DEG C, less than 12000g centrifugations
15 minutes.After centrifugation, mixture is separated into the colourless aqueous phase on red lower floor (phenol-chloroform phase), mesophase and upper strata, and RNA is deposited
It is water phase；
(4) water is mutually transferred to a new pipe, and mixing isopropanol is used for initial same to precipitate RNA from water phase, per 1 milliliter
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, it is centrifuged 10 minutes less than 12000g；
(5) supernatant is abandoned, RNA precipitate is washed once with 75% ethanol, per milliliter of TRIZOL examination for being used for initial homogeneity
Agent uses at least 1 milliliter 75% of ethanol, vortex mixed 2-8 DEG C, to be centrifuged 5 minutes less than 7500g；
(6) supernatant is abandoned, RNA precipitate (being air-dried or be vacuum dried 5-10 minutes) is simply dried, with 20-100ul without RNA
The water piping and druming of enzyme is dissolved several times RNA and is stored in -70 DEG C, in this process it may be noted that RNA precipitate should not be allowed to be completely dried, because
Its solubility will be substantially reduced for this.
The preparation of 3 standard DNA templates
To specifications, from esophageal squamous cell carcinoma with normal tissue in, using Invitrogen RTIZOL reagents
Box extracts total serum IgE, and further adoptsRNA clean-up kits (MACHEREY-NAGEL,
Germany post purifying) was carried out to total serum IgE, reverse transcription reaction was then carried out, reverse transcription system was：Reverse transcription random primer (2 μ
Mol/L) 1 μ L, the μ L of total serum IgE 4, the μ L of dNTP mixtures (every kind of 2.5mmol/L) 4,0.1mol/L DTT2 μ L, SuperScript
μ L (TAKARA) reaction conditions of RNase H reverse transcriptases (200U/uL) 2 are：37 DEG C of water-bath 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
The μ L of buffer10uL, dNTP Mixture (each 2.5mmol/L) 4, Sequence NO.4 (10pmol) 4 μ L, Sequence
The μ L of NO.5 (10pmol) 4, cDNA (0.1-2 μ g) 5 μ L, the μ L of Ex Taq archaeal dna polymerases 0.5, distilled water polishing to 100uL.Reaction
Condition is 94 DEG C of denaturations 5min；94 DEG C of denaturation 50s, 50 DEG C of annealing 50s, 72 DEG C extend 50s, 35cycles；Last 72 DEG C are prolonged
Stretch 10min.
5 μ L are sampled, row agarose gel electrophoresis detection is entered to the product of PCR amplifications, carry out cutting glue reclaim and purifying (reclaiming
Using kit：EZ-10Spin Column DNA Gel Extraction Kit), purified product is connected to into pGM-T clones
Carrier, in being subsequently transformed into DH5 α competent cells.Drawn by specificity of the sequence for SEQ ID NO.4 and SEQ ID NO.5
Thing screening positive clone.DNA is extracted after positive colony amplification, DNA adopts NanoDrop ND-1000 nucleic acid quantifications
Instrument quantitative (NanoDrop Technologies, Wihnington, Delaware) simultaneously does 10 times and is serially diluted and use as standard items
In the preparation of calibration curve, (standard DNA template concentration range is 108-102Copies/ μ l).
4 sensitivity experiments
Take recombinant plasmid and be diluted to 10 in proportion8、107、106、105、104、103、102Individual copy/μ L, carries out fluorescent quantitation
PCR, the detection sensitivity with the least concentration of test positive as the method.This research institute set up 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) is right
The total serum IgE reverse transcription synthesis cDNA of extraction, the kit includes gDNA Eraser DNase, can effectively remove the gene for mixing
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 it is as follows：
| Reagent | Usage amount |
| The reactant liquor of step 1 | 10.0μl |
| PrimeScript RT Enzyme Mix I | 1.0μl |
| RT Primer Mix | 1.0ul |
| 5×PrimeScript Buffer2 | 4μl |
| RNase Free dH2O | 4.0ul |
| Cumulative volume | 20ul |
37 DEG C of incubation 15min after above-mentioned component is well mixed, then 85 DEG C of inactivation 5sec, that is, obtain cDNA.
6 dye class fluorescence quantitative PCR detection HDESCC-lncRNA7 expressions
Using TAKARA SYBR Premix Ex TaqTMII (TIi RNaseH Plus) fluorescence quantitative kit (article No.
RR820A).50 μ L qRT-PCR reaction systems include：Upstream primer (10 μm of ol/L) 2 μ L；Downstream primer (10 μm of ol/L) 2 μ L；
Sample cDNA4 μ L；50×ROX Reference Dye1μL；2×SYBR Premex Ex Taq II(TIi RNaseH
Plus the μ L of) 25 μ L, plus ionized water to 50.Instrument adopts Applied Biosystems7500.Quantitative fluorescent PCR program：95℃
30s denaturations, 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-lncRNA7 in patients with esophageal squamous cell carcinoma tumor group
Knit and the expression in the tissue of knurl side.As a result it is as shown in Figure 3：QRT-PCR stable amplification results, wherein HDESCC-lncRNA7
Expression in the normal tissue is concentrated mainly on 0.002-0.05, hence it is evident that higher than tumor tissue and control plasmid 100copies,
HDESCC-lncRNA7 expressions are concentrated mainly on 0.000-0.003 in tumor tissue, these results suggest that HDESCC-lncRNA7
Expression is generally reduced in tumor tissues.According still further to relative expression quantity T-N>0 is defined as the index up-regulated；T-N<0 determines
Justice is lowered for the index expression.Then this experimental result shows：HDESCC-lncRNAl in 30 pairs of ESCC cancerous tissues, with pairing
Normal structure compares expression and lowers 22, according still further to formula：Lower expression number of cases/total detection number of cases x100% and define the index
Positive rate, preliminary identification then the index positive rate be 73.33%.
Embodiment 3qRT-PCR further verifies HDESCC-lncRNA7 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-lncRNA7qRT-PCR kit forms：
(1) upstream primer：SEQ ID NO.4
(2) downstream primer：SEQ ID NO.5；
Other reagents are with reference to SYBR Premix Ex TaqTMII (Tli RNaseH Plus) fluorescence quantitative kit
(Code No.RR820A)。
The detection of 2.HDESCC-lncRNA7qRT-PCR
The preparation of 2.1 total serum IgEs
The cancerous tissue of other 80 pairs of esophageal squamous cell carcinomas carninomatosis people is chosen and with normal tissue, according to Life
Technologies companiesReagent needed for reagent (article No. 15596026) and step extract total serum IgE, concrete ginseng
See specification.Again with NanoDrop ND-1000 nucleic acid quantifications instrument it is quantitative (NanoDrop Technologies, Wilmington,
The purity and concentration of the RNA for Delaware) quantitatively being extracted, denaturing formaldehyde gel electrophoresis quality inspection guarantees the integrality of the RNA for extracting.
2.2cDNA synthesis
Using TaKaRa kit PrimeScriptTM RT reagent Kit with gDNA Eraser(Perfect
Real Time) (article No. RR047A), the total serum IgE for detecting qualified is carried out into reverse transcription reaction.
2.3qRT-PCR detection
QRT-PCR adopts 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-lncRNA7 expressions.
3 testing results
Real-time quantitative PCR testing result shows, with pairing normal group in the sample of 80 couples of esophageal squamous cell carcinoma patients
Knit and compare, 65 pairs of patient indexs express substantially downward in tumor tissues, Positive rate is 81.25% (Fig. 4).We are right
Above-mentioned sample carries out being repeated 3 times qRT-PCR inspections, and as a result repeatability is up to 100%, shows the repeatability of kit of the present invention and steady
It is qualitative preferable.
Embodiment 4HDESCC-lncRNA7 is analyzed in the potential value of ESCC diagnosis and Index for diagnosis
HDESCC-lncRNA7 have detected on the basis of the expression of 110 ESCC patients, qRT-PCR is being reacted
As a result use SPSS For Windows20.0 softwares, related data to check through Normal test, select according to data type
Mann-Whitney inspections carry out data analysis, P<0.05 thinks that the differential expression of the index is statistically significant.QRT-PCR is tied
Expressions of the fruit analysis HDESCC-lncRNA7 in 110 pairs of ESCC samples substantially less than matches normal esophageal tissue (Fig. 5, P
<1×10-6).With going deep into for later stage result of study, functions and its mechanism of action of the HDESCC-lncRNA7 in ESCC will be by
Step is illustrated, and this novel long-chain non-coding RNA can not only become the related biomarker of diagnosis, more be expected to become new
ESCC therapy targets are improving, improve clinic ESCC therapeutic effects.Obviously, find it is more, more accurately as HDESCC-
It is expected to participate in the biomarker of auxiliary ESCC diagnosis, treatment and prognosis correlation as lncRNA7, with 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.
3. a kind of for esophageal squamous cell carcinoma auxiliary diagnosis or the real time quantitative PCR detecting reagent kit of outcome prediction, it is characterised in that
Design and synthesize out including the long-chain non-coding RNA that SEQ ID NO.1 are classified as according to nucleotides sequence and be specifically used for real-time quantitative
The detection primer of PCR；In specific primer such as sequence table shown in SEQ ID NO.4 and SEQ ID NO.5.
4. apply according to claim 1, the detection method of long-chain non-coding RNA includes extraction, the sample cDNA of sample RNA
Preparation, the amplification of lncRNA.
5. application according to claim 4, it is characterised in that the extraction of described sample RNA, the preparation of sample cDNA,
The concrete steps of the amplification of lncRNA include：
1) extraction of sample total serum IgE：Esophageal squamous cell is extracted according to reagent needed for the reagent of Life Technologies companies and step
The Total RNA of shape carcinoma tissues or tumour；The RNA's for quantitatively being extracted with NanoDrop ND-1000 nucleic acid quantification instrument again
Purity and concentration, denaturing formaldehyde gel electrophoresis quality inspection guarantees the integrality of the RNA for extracting；
2) preparation of sample cDNA：Using TaKaRa kit PrimeScriptTM RT reagent Kit with gDNA
Eraser synthesizes cDNA to the total serum IgE reverse transcription extracted；The kit includes RNase-Free DNase, can effectively remove and mix
Genomic DNA；
3) amplification of lncRNA：Using TAKARA SYBR Premix Ex TaqTM II fluorescence quantitative kits, with reverse transcription
CDNA carry out fluorescent quantitative PCR for template.
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| WO2012109476A2 (en) * | 2011-02-09 | 2012-08-16 | The University Of Rochester | Methods and compositions related to staufen 1 binding sites formed by duplexing alu elements |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012109476A2 (en) * | 2011-02-09 | 2012-08-16 | The University Of Rochester | Methods and compositions related to staufen 1 binding sites formed by duplexing alu elements |
| CN102433326A (en) * | 2011-04-25 | 2012-05-02 | 北京大学 | Long non-coding RNA and application thereof |
Non-Patent Citations (2)
| Title |
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| "Upregulation of the Long Non-coding RNA PlncRNA-1 Promotes Esophageal Squamous Carcinoma Cell Proliferation and Correlates with Advanced Clinical Stage";Chun-Mei Wang et al.;《Digestive Diseases and Sciences》;20131212;第591-597页 * |
| "长链非编码RNA SPRY4-IT1在食管鳞癌中的表达及对细胞生长的影响";谢海伟等;《中国肿瘤临床》;20130915;第40卷(第17期);摘要 * |
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| CN103952480A (en) | 2014-07-30 |
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