CN108707664A - A kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-COX19-2 and its application - Google Patents
A kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-COX19-2 and its application Download PDFInfo
- Publication number
- CN108707664A CN108707664A CN201810372418.3A CN201810372418A CN108707664A CN 108707664 A CN108707664 A CN 108707664A CN 201810372418 A CN201810372418 A CN 201810372418A CN 108707664 A CN108707664 A CN 108707664A
- Authority
- CN
- China
- Prior art keywords
- pancreas
- cancer
- prognosis
- cox19
- lnc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to cancer of pancreas prognosis molecule marker non-coding RNA Lnc-COX19-2 and its applications, it can effectively solve the problems, such as to prepare the kit for predicting Pancreas cancer patients prognosis, a kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-COX19-2, its transcript length is more than 200 nucleotide, and sequence is SEQ NO:1, the application in preparing cancer of pancreas prognosis real-time fluorescence quantitative PCR detection kit, the kit includes the specific primer for the kit expression for detecting Pancreas cancer patients prognosis being placed in box body, and primer sequence is SEQ No：2 and 3, the primer sequence of reference gene TUBA1A expression, primer sequence are SEQ No：4,5 and RNA is extracted from pancreatic tissue, and carry out the reagent of reverse transcription and quantitative fluorescent PCR.Molecular marker new as cancer of pancreas prognosis non-coding RNA Lnc-COX19-2 of the present invention can predict cancer of pancreas prognosis, and be treated in time to Pancreas cancer patients in time, extend the survival of patients time, improve survival rate, be the big innovation in cancer of pancreas prognosis.
Description
Technical field
The present invention relates to oncomolecularbiology, especially a kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-
COX19-2 and its application.
Background technology
Cancer of pancreas is malignant tumour relatively conventional in digestive system, and median survival time is less than six months, grade of malignancy
It is high, poor prognosis.In recent years, incidence at home and abroad has increased trend year by year.American Cancer Society's statistical data shows,
In the U.S., cancer of pancreas is the common cancer of incidence ranking the 4th, and 1 year and 5 years survival rate is 24% and 5% respectively.
China's pancreas cancer morbidity occupies the 7th in whole malignant tumours.Prediction in 2015 increases Pancreas cancer patients 100,000 newly.Cause
This, cancer of pancreas remains one of China's major cancers in recent years.The major way for the treatment of of pancreatic cancer is still operation excision at present.By
There is high malignancy in cancer of pancreas, local infiltration can occur in early days and golden body shifts, mostly oneself is in when most of sufferer is made a definite diagnosis
In late period, not as good as 20%, the survival rate for improving cancer of pancreas sufferer is not improved Resection Rate.Currently, not having also clinically
The risk for having ideal molecular indexes to assess prognosis and shift.Therefore, it is current to find new prognosis biomarker
Urgent problem.
Analysis result after the completion of human genome sequencing shows to encode the DNA of albumen in whole mankind's genomic DNA only
Account for a little, however coding protein, people were not once thinking that this part DNA was " rubbish once to overwhelming majority DNA
DNA".Recent studies indicate that these so-called junk DNAs, which can be also transcribed, generates RNA, and it is found that it is many this
RNA is not so-called transcription noise, although they cannot directly participate in gene code and albumen synthesis, but in gene
Transcriptional control, RNA shearings and modification etc. have the function of important, and vital work is played in many vital movements
With generation, diagnosis and treatment with disease have close relationship.The research of non-coding RNA is achieved not in recent years
Few achievement, but major part all concentrates on small non-coding RNA.Achievement in research about tiny RNA is also often cited as the world
Medicine breakthrough.However, the achievement in research for long-chain non-coding RNA is comparatively also seldom, belongs at present and grind
Study carefully than less clear one of transcription product.Long-chain non-coding RNA is the non-coding RNA that length is more than 200 nucleotide.Long-chain
The expression that non-coding RNA corresponds to gene is generally more relatively low than the expression of encoding egg white gene, this illustrates long-chain non-coding
RNA may primarily serve the effect of regulation and control.Recent studies indicate that long-chain non-coding RNA epigenetic regulation, transcription and
Gene expression regulation, transcriptional activation, transcription interference, cell differentiation regulation and control, cell cycle regulating on post-transcriptional level and agent
It plays an important role in numerous vital movements such as amount compensating effect.Lnc-COX19-2 is a kind of newfound non-coding
RNA, transcript length are more than 200 nucleotide.Currently, non-coding RNA Lnc-COX19-2 is in normal cell development or swells
The important function played during tumor occurrence and development is also in further research.Whether Lnc-COX19-2 can be used as one kind
New tumor markers are for predicting Pancreas cancer patients prognosis so far there are no open report.
Invention content
For the above situation, to overcome the defect of the prior art, the purpose of the present invention to be just to provide a kind of cancer of pancreas prognosis
Molecular marker non-coding RNA Lnc-COX19-2 and its application can effectively solve the examination for preparing prediction Pancreas cancer patients prognosis
The problem of agent box.
The technical solution that the present invention solves is a kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-COX19-2,
Its transcript length is more than 200 nucleotide, and sequence is SEQ NO:1；
The cancer of pancreas prognosis molecule marker non-coding RNA Lnc-COX19-2 is preparing cancer of pancreas prognosis real time fluorescent quantitative
Application in PCR detection kit；
The cancer of pancreas prognosis real-time fluorescence quantitative PCR detection kit includes being placed in suffering from for detecting cancer of pancreas in box body
The specific primer of the kit expression of person's prognosis, primer sequence are SEQ No：2 and 3, the primer of reference gene TUBA1A expression
Sequence, primer sequence are SEQ No：4,5 and RNA is extracted from pancreatic tissue, and carry out the examination of reverse transcription and quantitative fluorescent PCR
Agent.
Lnc-COX19-2 of the present invention is a kind of newfound non-coding RNA, can be effectively used for preparing Pancreas cancer patients prognosis
Kit, molecular marker new as cancer of pancreas prognosis non-coding RNA Lnc-COX19-2 can predict that cancer of pancreas is pre- in time
Afterwards, and in time Pancreas cancer patients are treated, extends the survival of patients time, improved survival rate, be one in cancer of pancreas prognosis
Big innovation, economic and social benefit are huge.
Description of the drawings
Fig. 1 is that real-time fluorescence quantitative PCR of the present invention analysis Lnc-COX19-2 is poor in normal structure and the expression in cancer of pancreas
Different figure.
Fig. 2 is the Lnc- that the present invention analyzes the influence prognosis survivorship curve of Pancreas cancer patients in Pancreatic Adenocarcinoma source
COX19-2 expresses high-low graph.
Specific implementation mode
It elaborates to the specific implementation mode of the present invention below in conjunction with concrete condition.
In specific implementation, a kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-COX19-2, is new to the present invention
It was found that a kind of non-coding RNA Lnc-COX19-2 molecular markers, be long-chain non-coding RNA, transcript length be more than 200
A nucleotide, the nucleotides sequence are classified as SEQ No：1：
SEQ NO:1
GACCCGCCTCACTAAGACCCGCCTCACTACGGACCCGCCCCACTACAGACCCGCCCCACTAAGACCCACCTCA
CTAAGACCCGCCTCACTACAGACCCGCCCCACTACGGACCCGCCCCACTATGGACCCTCCTCACTGCAGACCTGTTC
GCTCAAGTGGAACAGGCCGGTCCGGGCCCCATCTGAGGCTGCGAGGGCCGCGGAGATACGGATGCTGGTCCCAGGTG
CCTTCGGAGCTGGCCTAAGGTTCCACCCTGGCCCTCCCACCAGACCCCGTGTGACACAGCCTCCAGCCCTCTGTCCC
CTGGGGGCCCTTCAGCTCTGGTATTCAGTCAGAAATCCTAACACACCTTTCTTTTGTTTATTTATATTCTTTAGTTT
TGTGCACACTTTGAGGAATTGATTTAGGACAGGTTCATACTGAAAAAAACCTCAGCTGATGTTATCTGTGGGGGCTG
GGGAGGGTGTCAGGGACATTTGGTGGCTGAGGAGAGCGCGTCACTGCTATTGAATAGCTCCATTTAACACCAGCCAT
GTCTCCGCGTCTCAGGCACTTCTGTGAAATGTTCTCAGAACCCTGTGGTGACTGCGGCACACCCGGCAGGCCTTGCT
AGCACACGCCGCCCACTGGCAGGGCCCGGCCACCCTGGCTGTTGCCATTCTTTCGTAGGGTTTTGTTCATTTTACTA
TTTGTCATTTTTCTAGGAAACATCTGTTTTTGTAAAACAAACAAGGGGGAATCAAGTATTTTAACCACAAAGTATAA
ATACTGGCTCTAAGCTTTCATCACTTCATTGACAAACTGAATGCTGAGGAGGCTGAAGGCGAGGAGGCTTTTGCGGA
TGTGGACCTTGAGCTGCGTGTTTGGGAGACGCACAGGCCTCGGGGAGACTCAAGCCAGATGCCAGCCACGGGGACCA
CAGCCTTGGCCCAAGAGTGGCATTTGTGGGAAAAAGGAAACTTCAAGAAAAACACCCCTCCTGCTTCTCTTCCAGCA
GCTCGTCTCCTTCTGGAAGGATGTGGGGAGCGTCCAGGGTAACTAAGTGCTCTGCGTCCACCAGAGTCACGTCTCCC
CAGGAGGGCTGGGCCGCCTACGCCAGCACCTGCTGCACCCCCATGTGGTCCGGGGAGCAGTGCCGGTCCCCGCAGGG
CAGCTTTTTCATCAGCCGTTGGAGCTTGCTGGGGAAGCTCTGGTTCATGTAGCGGTAGAGAAGTGGTGTCACAAAGC
TGCTGGAGAAGGCCAGGAGTTTGGAGAAATCCTTCACAAAGTGCAGTAGCCCCAGGTAGTGTGCGTCCACGGGCTTC
CCTCGCGAGATGATGACCGTGTGCCCCAGCAGGATCAGATAGTGTGGCGTCCAGAGCCCAAACTGCGTGCACACGGT
GGCCACCAGCAGCCTGTGTGCCGAGGGCTCCAGCCGGCCCGTGTCCCGGTCCAGGGGCGTGTCCTCCCTGCGGACGC
GGGAGAGTAGCACCAGCGCGTAGAGGGTGGCCAGTGCTGGCACCACGTAGCCGATGAACACCAGCGTGGCGTCGGCA
GCTTCTGCGTTCTGCATCTTGGCGCACTCTAGCGCGCGGGTGGACACATGGCTGCAGATGTAGAAGAGCAGCGAGGA
GAAGCTGGTCAGCAGCGCGCCACCCCACACGAAGCCGCACACGTGCCGCGTGTTGTACACGCTGGCCATGTAGGTCC
GCGGCAGTGCACGCTCGATGTAGTGGTCGAGGCTCAGCAGGGCGGTGGAGTACATGGCCACCAGTGAGGACACATTG
AAGGGGATCTGCAGTGCCACGTGGACTTCGCCGCCCACACTCCACAGCGCCCACCGGGAGCTCGGGGGGCCGAGCAG
GTGCACAGGGGCCAGGGCGCTGAGCACCAGGCCTGCCACTGCCATGTTGACAAAGTACACGTCCGGCATGGTCATGC
TGGCCTTGCTGTGTAGGTTGGCCAGCACCAGCAGGGCGTTGTAGCACAGGCAGCTCCTCCACCAGCCCTGTGCCGTT
GAACCAGCTGCAGCTCCACATGGCGGCCGGCGAGGCTCCGCGACCCTGCGGAAAGAAGGAACAGGTGGTCAGCTCGC
GTCCCGTCCCAAAGCCCTGGCCCAGAAGTGCTGCAGGTACAGGGGTGCCTTCTCCTGGTGCAGTAATCGCTGCAGAC
CACAGAGCGAGGCTGCCTTTGGTGCATTTCTGTGAAAGTGAAGTTGAGCGTTGAAAAATGGCCGTCTCGGGTCGTGA
CCGGGG 2235；
The cancer of pancreas prognosis molecule marker non-coding RNA Lnc-COX19-2 is preparing cancer of pancreas prognosis real time fluorescent quantitative
Application in PCR detection kit；
The cancer of pancreas prognosis real-time fluorescence quantitative PCR detection kit includes being placed in suffering from for detecting cancer of pancreas in box body
The specific primer of the kit expression of person's prognosis, primer sequence are SEQ No：2 and 3, the primer of reference gene TUBA1A expression
Sequence, primer sequence are SEQ No：4,5 and RNA is extracted from pancreatic tissue, and carry out the examination of reverse transcription and quantitative fluorescent PCR
Agent, wherein：
SEQ NO:2
GTGGTGTCACAAAGCTGCTG
SEQ NO:3
GAGCCTCGACCACTACATCG
SEQ NO:4
aaaaggggtgggggagagat
SEQ NO:5
aatctctcccccaccccttt
The kit of Lnc-COX19-2 expression quantity is that real-time fluorescence determines PCR detection kit in the detection Pancreatic Adenocarcinoma.
The real-time fluorescence quantitative PCR detection kit includes the specific primer for carrying out real-time fluorescence quantitative PCR, packet
The specific primer for detecting Lnc-COX19-2 expression is included, primer sequence is SEQ NO:2,3 and reference gene
The primer sequence of TUBA1A expression, primer sequence are SEQ NO: 4,5.
The real-time fluorescence quantitative PCR detection kit also contains in addition to the primer of Lnc-COX19-2 from cancer of pancreas
RNA is extracted in tissue and carries out all reagents of reverse transcription and quantitative fluorescent PCR.Including：
(1) the extracted total RNA agents useful for same from the tumour of cancer of pancreas or normal control tissue, including Trizol liquid, chloroform, isopropyl
Alcohol inhibits RNA degradations solvent, 75% ethyl alcohol, DEPC water；
(2) it is cDNA agents useful for same by Lnc-COX19-2 reverse transcriptions by template of total serum IgE, including reverse transcription reaction buffer solution, contains
Have and contains Mg2+Triphosphoric acid base deoxynucleotide dNTP, reverse transcriptase M-MLV and random primer and Oligo dT primers it is mixed
Close object；
(3) by cDNA real-time quantitative PCR agents useful for same, including Lnc-COX19-2 real-time fluorescence quantitative PCRs specific primer,
TUBA1A internal references Specific PCR primers, real time fluorescent quantitative SYBR dyestuffs, the water without RNA enzyme.
The present invention by quantitative fluorescent PCR and survivorship curve analysis find the Lnc-COX19-2 in Pancreatic Adenocarcinoma source with
The survival rate of patient is related, and content is higher, and survival rate is higher.This method provides to predict that the prognosis life cycle of Pancreas cancer patients is analyzed
Strong technical support helps to improve the postoperative life quality of Pancreas cancer patients, works out aftertreatment scheme, improves life
Rate is deposited, there is far-reaching clinical meaning.And through site test, very satisfied advantageous effects are achieved, relevant information is such as
Under：
The kit that the reagent of the preparation detection Lnc-COX19-2 expression quantity of embodiment 1 is used to prepare Pancreas cancer patients prognosis (is used
In 30 secondary responses)
1. Trizo l20ml
2. inhibiting RNA degradation solvents 40ml
3. chloroform 80ml
4. isopropanol 80ml
5. DEPC water 10ml
6. the 100 μ l of mixture of 10 × random primer and Oligo dT primers
7. 5 × reverse transcription reaction buffer solution, 150 μ l
8. 10mM contains Mg2+100 μ l of triphosphoric acid base deoxynucleotide dNTP
9. 50 μ l of 200U/ μ l M-MLV reverse transcriptases
10. SYBR Green qPCR Mix 500μl
11. 3 μM of target gene Lnc-COX19-2 specific primers（Its sequence such as SEQ NO:2, shown in 3） 50μl
12. 3 μM of reference gene TUBA1A specific primers（Its sequence such as SEQ NO:4, shown in 5） 50μl
The detection of 2 tissue samples Lnc-COX19-2 of embodiment
1, cancer of pancreas to be measured or normal control tissue are collected, after physiological saline cleans up, is put into and fills inhibition RNA degradation solvents
Cryopreservation tube in, put spare to -80 DEG C of refrigerators.
2, the extracting of RNA in organizing：
（1）Liquid nitrogen is first added in mortar, then tissue is cut into small pieces and is clayed into power in liquid nitrogen, is taken with the spoon of Liquid nitrogen precooler
100mg organizes powder to be added in the EP pipes for the Trizol liquid for having filled 1ml.Organize total powder volume no more than Trizol used
The 10% of volume is sufficiently mixed uniformly.
（2）5 minutes are placed at room temperature for, the chloroform of 200 μ L is then added, EP is covered tightly and manages and acutely sway 0.5 minute.12000
Rev/min centrifugation 10 minutes.
（3）Take upper strata aqueous phase in a new EP pipes（The intermediate beds of precipitation and subnatant are not mixed into）, 500 μ L isopropyls are added
Alcohol mildly overturns mixing.10 minutes are placed at room temperature for, 12000 revs/min centrifuge 10 minutes.
（4）Liquid is carefully discarded supernatant, is added 75% ethyl alcohol of 1ml, vortex mixing, 12000 revs/min of centrifugations 5 at 4 DEG C
Minute.Repetitive operation is primary.
（5）Discard supernatant liquid（Residual liquid is removed as possible）, room temperature or vacuum drying 5~10 minutes（It is careful not to do
It is dry excessive, it otherwise can reduce the solubility of RNA）.RNA is dissolved with 50 μ L DEPC processed water.
（6）RNA concentration mensurations：It is measured with nucleic acid concentration analyzer, inhales 1 μ L RNA samples and add to sample well, according to
Reading directly determines the concentration of RNA.The OD260/OD280 ratios of the measurement of nucleic acid concentration analyzer, ratio is between 1.8-2.0
Think that RNA purity is fine.Finally, it is spare to be stored in -80 DEG C of refrigerators by RNA.
3, the reverse transcription of Lnc-COX19-2 RNA：Use the reverse transcription reagents of the green skies Bioisystech Co., Ltd in Shanghai
Box（D7170S）.It is as follows.
（1）Remove genomic DNA：Template Total RNA, 5 × gDNA Eraser Buffer are thawed on ice, 5 ×
RT Buffer, 10 × RT Primer Mix, DEPC-treated Water are in (15-25 DEG C) defrosting of room temperature, after defrosting rapidly
It is placed on ice.Using it is preceding by each solution mixing and it is of short duration centrifugation so that all liq is settled down to tube bottom.The denaturation of RNA,
RNA sample thermal denaturation under the conditions of 65 DEG C is immediately placed in ice water cooling after 5 minutes.According to the form below ingredient is in preparation reaction on ice
Then mixed liquor is dispensed into each reaction tube, is eventually adding RNA sample again.
| Reagent | Usage amount |
| 5×gDNA Eraser Buffer | 2μl |
| Total RNA | up to 0.5μg |
| DEPC-treated Water | To 10 μ l |
| Total volume | 10μl |
（2）In PCR instrument or in water-bath, 37 DEG C are incubated 2 minutes.Be immediately placed in place on ice it is spare.
（3）Reverse transcription system is prepared：Reaction solution preparation is carried out on ice, and reverse transcription reaction is carried out immediately after soft mixing.
Mixed liquor is prepared according to the reverse transcription reaction system of following table.
| Reagent | Usage amount |
| 5×RT Buffer（Containing Mg2+And dNTP） | 4μl |
| 10×RT Primer Mix（Oligo dT Primer and Random Hexamers mixtures） | 2μl |
| BeyoRT II M-MLV reverse transcriptases (RNase H-)（Inhibitor containing RNase） | 2μl |
| DEPC-treated water | 2μl |
| Total RNA after above-mentioned removal gDNA | 10μl |
| Total volume | 20μl |
（4）42 DEG C are incubated 60 minutes to carry out reverse transcription reaction, after subsequent 80 DEG C are incubated 10 minutes inactivation reverse transcriptases
It is put on ice.For the template of secondary structure complexity or high GC content, reverse transcription temperature can be improved to 50 DEG C, reversed with enhancing
Record efficiency.
（5）Obtained cDNA can immediately or -80 DEG C freeze after be used for follow-up real-time fluorescence quantitative PCR, cDNA preferably avoided
More multigelations.
4, real-time quantitative PCR is carried out using the specific primer of Lnc-COX19-2：Specific primer is in raw work bioengineering
（Shanghai）Limited liability company synthesizes, and includes the specific primer for detecting Lnc-COX19-2 expression, primer sequence SEQ
NO:2, the primer sequence that 3 and reference gene TUBA1A is expressed, primer sequence are SEQ NO: 4,5.Real-time quantitative PCR its
Its reagent utilizes the BeyoFast SYBR Green qPCR Mix (2 ×) of the green skies Bioisystech Co., Ltd in Shanghai, tool
Steps are as follows for body.
（1）Melt and mixing PCR reacts required various solution, BeyoFast SYBR Green qPCR Mix are complete
Melt and mixing is placed in ice chest.
（2）PCR reaction systems (by taking 96 orifice plates as an example) are set on ice bath：
| Reagent | Usage amount |
| BeyoFast™ SYBR Green qPCR Mix (2X, Low ROX) | 10μl |
| Specific primer (3 μM of concentration) | 2μl |
| Template DNA | 2μl |
| Water without RNA enzyme | 6μl |
| Total volume | 20μl |
(3) the usually amount of DNA profiling with 1-10ng cDNA is with reference to dosage, and final concentration of 0.2-0.5 μM of primer when can be obtained
Good detection result is obtained, the final concentration of primer also can be according to circumstances adjusted.If it is necessary, gradient dilution can be carried out to template,
With the template usage amount that determination is best.When reverse transcription PCR cDNA obtained by the reaction is directly as template, additive amount does not exceed
PCR reacts the 10% of total volume.The recommendation response system of 96 orifice plates is 20 μ l, can also in proportion be expanded according to actual experiment demand
Big or diminution reaction system.
(4) mixing or slight Vortex mixings are gently blown and beaten with pipettor, room temperature centrifuges the several seconds, liquid is made to accumulate in pipe
Bottom.
(5) the PCR reaction tubes set or PCR reaction plates are placed on fluorescence quantitative PCR instrument, start PCR reactions.
(6) PCR response procedures：The pre-degeneration that template is carried out before real-time fluorescence quantitative PCR reacts, is typically set at 95 DEG C
2 minutes.Using following PCR programs, this program is by taking ABI 7900HT fluorescence quantitative PCR instruments as an example：
A. pre-degeneration：95℃ 2min
B. it is denaturalized：95℃ 15sec
C. annealing/extension：60℃ 15-30sec
D. step b and step c is repeated, in total 40 cycles
E. melting curve analysis (optional)：95℃ 15sec, 60℃ 15sec, 95℃ 15sec
F. the software analysis result for using fluorescence quantitative PCR instrument to provide
Three-step approach only need to after annealing/extension plus 72 DEG C of 30sec of a step, then repeat step b, c and it is increased the step for it is total
40 cycles.
5, the data analysis of Lnc-COX19-2 expression quantity：This experimental data is included in 60 Pancreas cancer patients and its normal right
According to tissue.The interpretation of result of real-time quantitative PCR uses relative quantitation method, that is, 2^-△△CtMethod.It is specific as follows：It first, will be once real
All gene C t values tested are put in order, are subtracted later with the Ct values of the target gene Lnc-COX19-2 in each group of tumor sample
The Ct values of itself reference gene TUBA1A, obtained number are exactly △ Ct；Changing formula into is exactly：△Ct=Ct（Target gene Lnc-
COX19-2）-Ct（Reference gene TUBA1A）；Then, by each group of each target gene of tumor sample Lnc-COX19-2's
△ Ct are calculated.The △ Ct of normal control tissue group sample are subtracted with the △ Ct of tumor tissues sample in this experiment, and simultaneously
Opposite number is taken to all results, the result which obtains is exactly-△ △ Ct.Finally, p- △ △ Ct carry out 2 power operation,
That is 2^-△△CtJust show that the multiple of expression quantity changes.In triplicate, it is examined using nonparametric t- for statistical analysis.We send out
Existing, the expression quantity of Lnc-COX19-2 is higher than normal control in the tumor tissues of cancer of pancreas（See Fig. 1）, difference have statistics it is poor
Different (p< 0.05).
6, by 60 Pancreas cancer patients follow-up statistics being included in above-mentioned experiment, including patient's First episode
Time, treatment, recurrence status and death time etc., follow up time are at least 12 months.In selected Pancreas cancer patients
In, the expression value for choosing fluorescence real-time quantitative PCR analysis is reference standard, and the corresponding normal structure of acquired results is compared
Compared with.Lnc-COX19-2 expression is defined as Lnc-COX19-2 high expression groups higher than the patient of normal control tissue in tumor tissues,
Remaining is low expression group.By Kaplan-Meier survival analysis, Lnc-COX19-2 high expresses the life cycle ratio Lnc- of patient
The patient of COX19-2 low expression groups is substantially reduced, and prognosis is worse（See Fig. 2）, difference have it is statistically significant (p< 0.05).
Therefore, Lnc-COX19-2 can be used as the specificity molecular marker of Pancreas cancer patients prognosis.
By above it should be apparent that the invention discloses a kind of new cancer of pancreas prognosis molecule marker non-codings
RNA Lnc-COX19-2 are used to prepare the kit of Pancreas cancer patients, the Lnc- tissue-derived by detecting pancreatic tumour
The kit of COX19-2.Lnc-CIT-1 up-regulated expressions in cancer of pancreas, the pancreas of height expression Lnc-COX19-2 are confirmed by research
Adenocarcinoma patients, overall survival are worse.Therefore, pass through the expression of Lnc-COX19-2 in detection Pancreas cancer patients tumor tissues
Level can make Pancreas cancer patients prognosis inspection diagnosis, and accuracy rate is up to 96% or more, and is treated in time, improves and suffers from
Person's survival rate and life quality have far-reaching clinical application significance and application value, economic and social benefit huge.
Sequence table
<110>The first affiliated hospital of Zhengzhou University
<120>A kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-COX19-2 and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2235
<212> DNA
<213> Homo sapiens
<400> 1
gacccgcctc actaagaccc gcctcactac ggacccgccc cactacagac ccgccccact 60
aagacccacc tcactaagac ccgcctcact acagacccgc cccactacgg acccgcccca 120
ctatggaccc tcctcactgc agacctgttc gctcaagtgg aacaggccgg tccgggcccc 180
atctgaggct gcgagggccg cggagatacg gatgctggtc ccaggtgcct tcggagctgg 240
cctaaggttc caccctggcc ctcccaccag accccgtgtg acacagcctc cagccctctg 300
tcccctgggg gcccttcagc tctggtattc agtcagaaat cctaacacac ctttcttttg 360
tttatttata ttctttagtt ttgtgcacac tttgaggaat tgatttagga caggttcata 420
ctgaaaaaaa cctcagctga tgttatctgt gggggctggg gagggtgtca gggacatttg 480
gtggctgagg agagcgcgtc actgctattg aatagctcca tttaacacca gccatgtctc 540
cgcgtctcag gcacttctgt gaaatgttct cagaaccctg tggtgactgc ggcacacccg 600
gcaggccttg ctagcacacg ccgcccactg gcagggcccg gccaccctgg ctgttgccat 660
tctttcgtag ggttttgttc attttactat ttgtcatttt tctaggaaac atctgttttt 720
gtaaaacaaa caagggggaa tcaagtattt taaccacaaa gtataaatac tggctctaag 780
ctttcatcac ttcattgaca aactgaatgc tgaggaggct gaaggcgagg aggcttttgc 840
ggatgtggac cttgagctgc gtgtttggga gacgcacagg cctcggggag actcaagcca 900
gatgccagcc acggggacca cagccttggc ccaagagtgg catttgtggg aaaaaggaaa 960
cttcaagaaa aacacccctc ctgcttctct tccagcagct cgtctccttc tggaaggatg 1020
tggggagcgt ccagggtaac taagtgctct gcgtccacca gagtcacgtc tccccaggag 1080
ggctgggccg cctacgccag cacctgctgc acccccatgt ggtccgggga gcagtgccgg 1140
tccccgcagg gcagcttttt catcagccgt tggagcttgc tggggaagct ctggttcatg 1200
tagcggtaga gaagtggtgt cacaaagctg ctggagaagg ccaggagttt ggagaaatcc 1260
ttcacaaagt gcagtagccc caggtagtgt gcgtccacgg gcttccctcg cgagatgatg 1320
accgtgtgcc ccagcaggat cagatagtgt ggcgtccaga gcccaaactg cgtgcacacg 1380
gtggccacca gcagcctgtg tgccgagggc tccagccggc ccgtgtcccg gtccaggggc 1440
gtgtcctccc tgcggacgcg ggagagtagc accagcgcgt agagggtggc cagtgctggc 1500
accacgtagc cgatgaacac cagcgtggcg tcggcagctt ctgcgttctg catcttggcg 1560
cactctagcg cgcgggtgga cacatggctg cagatgtaga agagcagcga ggagaagctg 1620
gtcagcagcg cgccacccca cacgaagccg cacacgtgcc gcgtgttgta cacgctggcc 1680
atgtaggtcc gcggcagtgc acgctcgatg tagtggtcga ggctcagcag ggcggtggag 1740
tacatggcca ccagtgagga cacattgaag gggatctgca gtgccacgtg gacttcgccg 1800
cccacactcc acagcgccca ccgggagctc ggggggccga gcaggtgcac aggggccagg 1860
gcgctgagca ccaggcctgc cactgccatg ttgacaaagt acacgtccgg catggtcatg 1920
ctggccttgc tgtgtaggtt ggccagcacc agcagggcgt tgtagcacag gcagctcctc 1980
caccagccct gtgccgttga accagctgca gctccacatg gcggccggcg aggctccgcg 2040
accctgcgga aagaaggaac aggtggtcag ctcgcgtccc gtcccaaagc cctggcccag 2100
aagtgctgca ggtacagggg tgccttctcc tggtgcagta atcgctgcag accacagagc 2160
gaggctgcct ttggtgcatt tctgtgaaag tgaagttgag cgttgaaaaa tggccgtctc 2220
gggtcgtgac cgggg 2235
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 2
gtggtgtcac aaagctgctg 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 3
gagcctcgac cactacatcg 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 4
aaaaggggtg ggggagagat 20
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 5
aatctctccc ccaccccttt 20
Claims (3)
1. a kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-COX19-2 is long-chain non-coding RNA, transcript
Length is more than 200 nucleotide, which is classified as SEQ No：1.
2. cancer of pancreas prognosis molecule marker non-coding RNA Lnc-COX19-2 described in claim 1 is pre- in preparation cancer of pancreas
Application in real-time fluorescence quantitative PCR detection kit afterwards.
3. the cancer of pancreas prognosis molecule marker non-coding RNA Lnc-COX19-2 described in claim 2 is pre- in preparation cancer of pancreas
Application in real-time fluorescence quantitative PCR detection kit afterwards, which is characterized in that the cancer of pancreas prognosis real time fluorescent quantitative
PCR detection kit includes the specific primer for the kit expression for detecting Pancreas cancer patients prognosis being placed in box body,
Primer sequence is SEQ No：2 and 3, the primer sequence of reference gene TUBA1A expression, primer sequence are SEQ No：4,5 and from pancreas
RNA is extracted in glandular tissue, and carries out the reagent of reverse transcription and quantitative fluorescent PCR.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810372418.3A CN108707664A (en) | 2018-04-24 | 2018-04-24 | A kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-COX19-2 and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810372418.3A CN108707664A (en) | 2018-04-24 | 2018-04-24 | A kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-COX19-2 and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108707664A true CN108707664A (en) | 2018-10-26 |
Family
ID=63867400
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810372418.3A Pending CN108707664A (en) | 2018-04-24 | 2018-04-24 | A kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-COX19-2 and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108707664A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111118154A (en) * | 2020-01-16 | 2020-05-08 | 华南协同创新研究院 | Application of LINC01272 in preparation of tumor detection reagent and/or treatment drug |
| CN113249479A (en) * | 2021-05-24 | 2021-08-13 | 大连医科大学附属第一医院 | Pancreatic cancer related lncRNA marker, probe and application of detection kit in pancreatic cancer diagnosis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106350600A (en) * | 2016-11-04 | 2017-01-25 | 叶伟亮 | Application of LOC80054 in diagnosis or prognosis of pancreatic cancer |
| CN106399569A (en) * | 2016-12-01 | 2017-02-15 | 北京致成生物医学科技有限公司 | Application of C2lorf82 in preparation of pancreatic cancer prognosis evaluation products |
-
2018
- 2018-04-24 CN CN201810372418.3A patent/CN108707664A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106350600A (en) * | 2016-11-04 | 2017-01-25 | 叶伟亮 | Application of LOC80054 in diagnosis or prognosis of pancreatic cancer |
| CN106399569A (en) * | 2016-12-01 | 2017-02-15 | 北京致成生物医学科技有限公司 | Application of C2lorf82 in preparation of pancreatic cancer prognosis evaluation products |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111118154A (en) * | 2020-01-16 | 2020-05-08 | 华南协同创新研究院 | Application of LINC01272 in preparation of tumor detection reagent and/or treatment drug |
| CN111118154B (en) * | 2020-01-16 | 2022-10-18 | 华南协同创新研究院 | Application of LINC01272 in preparation of tumor detection reagent and/or treatment drug |
| CN113249479A (en) * | 2021-05-24 | 2021-08-13 | 大连医科大学附属第一医院 | Pancreatic cancer related lncRNA marker, probe and application of detection kit in pancreatic cancer diagnosis |
| CN113249479B (en) * | 2021-05-24 | 2022-05-20 | 大连医科大学附属第一医院 | Pancreatic cancer related lncRNA marker, probe and application of detection kit in pancreatic cancer diagnosis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5674682A (en) | Nucleic acid primers for detecting micrometastasis of prostate cancer | |
| CN109182521B (en) | Application of circRNA as thyroid papillary carcinoma marker | |
| CN109504780B (en) | DNA methylation qPCR kit for lung cancer detection and use method thereof | |
| Geng et al. | Correlation between TERT C228T and clinic-pathological features in pediatric papillary thyroid carcinoma | |
| CN107519193B (en) | Molecular diagnostic marker for early stage esophageal squamous carcinoma and application thereof | |
| CN108707664A (en) | A kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-COX19-2 and its application | |
| WO2022147991A1 (en) | Primer composition for detecting tert promoter methylation and use thereof | |
| CN113025692B (en) | Primate DNA methylation relative quantification kit | |
| CN110157808A (en) | A kind of application of non-coding RNA relevant to larynx squamous carcinoma occurrence and development | |
| CN105802969A (en) | lncRNA ENST00000581794.1 and preparation, diagnostic agent, medicine or kit and application | |
| CN108424965A (en) | A kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-CRYBA4-23 and its application | |
| CN108546760A (en) | A kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-COL20A1-2 and its application | |
| CN108728547A (en) | Application, kit and the detection method of kidney prognosis novel molecular marker non-coding RNA LINC00158 | |
| CN109266742A (en) | Predict the biomarker long-chain non-coding RNA LINC02456 and kit of colon cancer prognosis | |
| CN108753978A (en) | Application, kit and the detection method of lung cancer for prognosis novel molecular marker non-coding RNA LINC01124 | |
| CN108998519A (en) | Predict the biomarker long-chain non-coding RNA LINC02413 and kit of colon cancer prognosis | |
| CN108753960A (en) | A kind of gastric cancer prognosis molecule marker non-coding RNA Lnc-DENR-2 and its application | |
| CN108546759A (en) | A kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-CPM-2 and its application | |
| CN108753977A (en) | Application, kit and the detection method of lung cancer for prognosis novel molecular marker non-coding RNA LINC00958 | |
| CN108486253A (en) | A kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-DCTD-25 and its application | |
| CN108796087A (en) | Application, kit and the detection method of kidney prognosis novel molecular marker non-coding RNA LINC00668 | |
| CN108424964A (en) | A kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-CLEC18B-3 and its application | |
| CN108642183A (en) | Application, kit and the detection method of kidney prognosis novel molecular marker non-coding RNA LINC00671 | |
| CN108728546A (en) | Application, kit and the detection method of lung cancer for prognosis novel molecular marker non-coding RNA LINC00963 | |
| AU2004224448A1 (en) | Detection and monitoring of lung cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20181026 |