CN109234365A - A method of detection miR5100, CXCL-12 and IL-8 relative expression quantity - Google Patents
A method of detection miR5100, CXCL-12 and IL-8 relative expression quantity Download PDFInfo
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- CN109234365A CN109234365A CN201811348833.1A CN201811348833A CN109234365A CN 109234365 A CN109234365 A CN 109234365A CN 201811348833 A CN201811348833 A CN 201811348833A CN 109234365 A CN109234365 A CN 109234365A
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- cxcl
- mir5100
- denaturation
- expression quantity
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- 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
- C12Q1/6851—Quantitative amplification
Abstract
The invention proposes a kind of methods for detecting miR5100, CXCL-12 and IL-8 relative expression quantity, and with qPCR technology, the relative expression quantity of a indexs more to miR5100, CXCL-12 and IL-8 is detected；Method proposed by the present invention only needs to use blood as sample, and entire detection process cost is lower；Method proposed by the present invention is more convenient relative to conventional method, and result is faster out；Relative to the method having disclosed, result is more credible.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of detection miR5100, CXCL-12 and IL-8 relative expression
The method of amount.
Background technique
MiRNA is a kind of single stranded RNA of non-coding, and length is about 18-25 nucleotide.It can combine target gene
On the 3 ' UTR of mRNA, mRNA is caused to degrade or inhibit its translation, to regulate and control the expression of target gene, participates in cell Proliferation, divides
A series of important biological processes such as change, apoptosis, realize the regulation of post-transcriptional level.
Chemokines CC XCL-12 is also known as the cell factor that stromal cell derived factor-1 (SDF-1) is small molecule, is one
It is positioned at the CXC class chemotactic factor (CF) that No. 10 chromosomes are generated by stroma cell, belongs to chemotactic protein family, Chemokines CC XCL-
12 pairs of lymphocytes have strong chemotaxis and play an important role in development, it is now recognized that CXCR-4 and CXCR-7 are
Two receptors of CXCL-12.
Interleukin 8 (IL-8), also known as Chemokines CC XCL-8 are the secretions such as macrophage and epithelial cell
Cell factor, IL-8 are a kind of most important neutrophil activation chemotactic factor (CF)s, and neutrophil leucocyte and lymph can be promoted thin
The exudation and aggregation of born of the same parents, and different cells is acted on, promote inflammatory reaction process, vasostimulant formation, promote have silk point
Split, adjust host immune function etc..
The RNA that the detection method of existing miR5100, CXCL-12 and IL-8 are all based on purifying is template, due to extracting
RNA precipitate and recycling is incomplete in nucleic acid, it will some RNA is inevitably caused to lose.In addition, RNA extraction process
In, easily cause pollution and waste.
As it can be seen that there are also to be improved for the prior art.
Summary of the invention
In view of this, present invention application qPCR technology, by the opposite of the more a indexs of miR5100, CXCL-12 and IL-8
Expression quantity is detected, and more convenient relative to conventional method, result is more very fast out；Relative to the method having disclosed, result
It is more credible.
The technical scheme of the present invention is realized as follows: the present invention provides a kind of detection miR5100, CXCL-12 and
The method of IL-8 relative expression quantity, which comprises the extraction of total serum IgE, RNA reverse transcription reaction and real-time fluorescence in sample
Quantitative PCR.
On the basis of above technical scheme, it is preferred that the extraction of total serum IgE in the sample is using kit
RNAiso Plus, comprising the following steps:
(1) subject's blood 4ml is extracted, 4 DEG C of placements are spare；
(2) 1ml RNAiso Plus is added into 200ul whole blood, cracks 10min on ice；
(3) chloroform of 200ul is added, is uniformly mixed, stands 5min on ice；
(4) 4 DEG C of 12000g/min are centrifuged 10min, collect supernatant in the 1.5ml centrifuge tube of no RNA enzyme；
(5) it is added into centrifuge tube and stands 10min on ice with the isometric isopropanol of supernatant, mixing；
(6) 4 DEG C of 12000g/min are centrifuged 10min, abandon supernatant；
(7) 1ml water-reducible 75% ethyl alcohol of DEPC is added, 7500g/min is centrifuged 2min；
(8) supernatant, dry 5min are abandoned；
(9) add 20ul DEPC water room-temperature dissolution；
(10) RNA concentration is measured using Nanodrop 2000, retains OD260/OD280The sample being worth between 1.8-2.0.
On the basis of above technical scheme, it is preferred that the RNA reverse transcription reaction is using kit
PrimeScript RT reagent Kit with gDNA Eraser, includes the following steps:
(1) the removal reaction of genomic DNA
5x gDNA Eraser Buffer 2.0ul, gDNA Eraser 1.0ul, Total RNA are added in micro tube
1.0ug、RNase Free dH2O supplies 10ul, and 42 DEG C of removal genomic DNAs react 2min；
(2) reverse transcription reaction
Reaction system is 20ul: RNase Free dH is separately added into the micro tube of step 12O 4ul、5x
Added after PrimeScript Buffer (for Real Time) 4.0ul, mixing RT Primer Mix 1.0ul,
PrimeScript RT Enzyme Mix I 1.0ul is mixed gently.Specifically, reaction condition are as follows: 37 DEG C of reverse transcription 15min,
85 DEG C of denaturation 5s, 4 DEG C it is stored refrigerated, wherein the cDNA of synthesis needs to save at -20 DEG C.
On the basis of above technical scheme, it is preferred that the RNA reverse transcription reaction condition are as follows: 37 DEG C of reverse transcription reactions
Time is 30min.
On the basis of above technical scheme, it is preferred that the real-time fluorescence quantitative PCR, including miR5100 is carried out
Real-time fluorescence quantitative PCR carries out real-time fluorescence quantitative PCR to CXCL-12 and carries out real-time fluorescence quantitative PCR to IL-8.
It is further preferred that described carry out real-time fluorescence quantitative PCR to miR5100, used kit is Hieff
QPCR SYBR Green Master Mix (No Rox), includes the following steps:
(1) 20ul qPCR reaction system: 2X SYBR Green Mix 10ul, cDNA 2ul, U6 upstream primer 0.8ul,
U6 downstream primer 0.8ul, ddH2O supplies 20ul；
(2) qPCR reaction condition are as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 10s, 60 DEG C of annealing 20s, 72 DEG C of extensions
20s, totally 40 recycle；95 DEG C of denaturation 15s of solubility curve, 60 DEG C of renaturation 60s, 95 DEG C of denaturation 15s, U6 is as miR5100 internal reference.
It is further preferred that described carry out real-time fluorescence quantitative PCR to CXCL-12, used kit is Hieff
QPCR SYBR Green Master Mix (No Rox), includes the following steps:
(1) 20ul qPCR reaction system: 2X SYBR Green Mix 10ul, cDNA2ul, GAPDH upstream primer
0.8ul, GAPDH downstream primer 0.8ul, ddH2O supplies 20ul；
(2) qPCR reaction condition are as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 10s, 60 DEG C of annealing 20s, 72 DEG C of extensions
20s, totally 40 recycle.95 DEG C of denaturation 15s of solubility curve, 60 DEG C of renaturation 60s, 95 DEG C of denaturation 15s.GAPDH is as in CXCL-12
Ginseng.
It is further preferred that described carry out real-time fluorescence quantitative PCR to IL-8, used kit is Hieff qPCR
SYBR Green Master Mix (No Rox), includes the following steps:
(1) 20ul qPCR reaction system: 2X SYBR Green Mix 10ul, cDNA2ul, GAPDH upstream primer
0.8ul, GAPDH downstream primer 0.8ul, ddH2O supplies 20ul；
(2) qPCR reaction condition are as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 10s, 60 DEG C of annealing 20s, 72 DEG C of extension 20s,
Totally 40 circulations.95 DEG C of denaturation 15s of solubility curve, 60 DEG C of renaturation 60s, 95 DEG C of denaturation 15s.Internal reference of the GAPDH as IL-8.
A kind of method of detection miR5100, CXCL-12 and IL-8 relative expression quantity of the invention has compared with the existing technology
Have it is following the utility model has the advantages that
(1) present invention by the relative expression quantity to the more a indexs of miR5100, CXCL-12 and downstream target gene IL-8 into
Row detection, more convenient relative to conventional method, result is faster out；Relative to the method having disclosed, result is more credible；
(2) method of a kind of detection miR5100, CXCL-12 and IL-8 relative expression quantity of the invention, it is only necessary to which blood is made
For sample, entire detection process is at low cost, and speed is fast, easy to operate.
Specific embodiment
Below in conjunction with embodiment of the present invention, the technical solution in embodiment of the present invention is carried out clearly and completely
Description, it is clear that described embodiment is only some embodiments of the invention, rather than whole embodiments.Base
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts all
Other embodiments shall fall within the protection scope of the present invention.
Embodiment one
A kind of method for extracting total serum IgE in sample is present embodiments provided, used kit is RNAiso Plus, specifically
Steps are as follows:
(1) subject's blood 4ml is extracted, 4 DEG C of placements are spare；
(2) 1ml RNAiso Plus is added into 200ul whole blood, cracks 10min on ice；
(3) 1/5 chloroform of 200ul is added, it is slowly reverse, 5min is stood on ice；
(4) 4 DEG C of 12000g/min are centrifuged 10min, collect 1.5ml centrifuge tube of the supernatant without RNA enzyme；
(5) it is added into centrifuge tube and stands 10min on ice with the isometric isopropanol of supernatant, mixing；
(6) 4 DEG C of 12000g/min are centrifuged 10min, abandon supernatant；
(7) 1ml water-reducible 75% ethyl alcohol of DEPC is added, 7500g/min is centrifuged 2min；
(8) supernatant, dry 5min are abandoned；
(9) add 20ul DEPC water room-temperature dissolution；
RNA concentration is measured using Nanodrop 2000, retains OD260/OD280The sample being worth between 1.8-2.0.
Embodiment two
A kind of RNA reverse transcription reaction is present embodiments provided, used kit is PrimeScript RT reagent
Kit with gDNAEraser, the specific steps are as follows:
(1) the removal reaction of genomic DNA
5x gDNA Eraser Buffer 2.0ul, gDNA Eraser 1.0ul, Total RNA are added in micro tube
1.0ug、RNase Free dH2O supplies 10ul, and 42 DEG C of removal genomic DNAs react 2min；
(2) reverse transcription reaction
Reaction system is 20ul: RNase Free dH is separately added into the micro tube of step 12O 4ul、 5x
Added after PrimeScript Buffer (for Real Time) 4.0ul, mixing RT Primer Mix1.0ul,
PrimeScript RT Enzyme Mix I 1.0ul is mixed gently.
Response procedures are as follows: 37 DEG C of reverse transcription 30min, 85 DEG C of denaturation 5s, 4 DEG C it is stored refrigerated, wherein the cDNA of synthesis needs
It is saved at -20 DEG C.
Embodiment three
Present embodiments provide it is a kind of quantitative method is carried out to miR5100 relative concentration with quantitative fluorescent PCR, used
Kit is Hieff qPCR SYBR Green Master Mix (No Rox), the specific steps are as follows:
(1) 20ul qPCR reaction system: 2X SYBR Green Mix 10ul, cDNA 2ul, U6 upstream primer 0.8ul,
U6 downstream primer 0.8ul, ddH2O supplies 20ul；
(2) qPCR reaction condition are as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 10s, 60 DEG C of annealing 20s, 72 DEG C of extensions
20s, totally 40 recycle；95 DEG C of denaturation 15s of solubility curve, 60 DEG C of renaturation 60s, 95 DEG C of denaturation 15s, U6 is as miR5100 internal reference.
Example IV
Present embodiments provide it is a kind of quantitative method is carried out to CXCL-12 relative concentration with quantitative fluorescent PCR, used
Kit is Hieff qPCR SYBR Green Master Mix (No Rox), is included the following steps:
(1) 20ul qPCR reaction system: 2X SYBR Green Mix 10ul, cDNA2ul, GAPDH upstream primer
0.8ul, GAPDH downstream primer 0.8ul, ddH2O supplies 20ul；
(2) qPCR reaction condition are as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 10s, 60 DEG C of annealing 20s, 72 DEG C of extensions
20s, totally 40 recycle.95 DEG C of denaturation 15s of solubility curve, 60 DEG C of renaturation 60s, 95 DEG C of denaturation 15s, GAPDH is as in CXCL-12
Ginseng.
Embodiment five
Present embodiments provide it is a kind of quantitative method is carried out to IL-8 relative concentration with quantitative fluorescent PCR, use examination
Agent box is Hieff qPCR SYBR Green Master Mix (No Rox), is included the following steps:
(1) 20ul qPCR reaction system: 2X SYBR Green Mix 10ul, cDNA2ul, GAPDH upstream primer
0.8ul, GAPDH downstream primer 0.8ul, ddH2O supplies 20ul；
(2) qPCR reaction condition are as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 10s, 60 DEG C of annealing 20s, 72 DEG C of extensions
20s, totally 40 recycle.95 DEG C of denaturation 15s of solubility curve, 60 DEG C of renaturation 60s, 95 DEG C of denaturation 15s, GAPDH is as the interior of IL-8
Ginseng.
The foregoing is merely better embodiments of the invention, are not intended to limit the invention, all of the invention
Within spirit and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of method for detecting miR5100, CXCL-12 and IL-8 relative expression quantity, it is characterised in that: the described method includes:
The extraction of total serum IgE, RNA reverse transcription reaction and real-time fluorescence quantitative PCR in sample.
2. a kind of method for detecting miR5100, CXCL-12 and IL-8 relative expression quantity as claimed in claim 1, feature
Be: the extraction of total serum IgE in the sample is RNAiso Plus using kit, comprising the following steps:
(1) subject's blood 4ml is extracted, 4 DEG C of placements are spare；
(2) 1ml RNAiso Plus is added into 200ul whole blood, cracks 10min on ice；
(3) chloroform of 200ul is added, is uniformly mixed, stands 5min on ice；
(4) 4 DEG C of 12000g/min are centrifuged 10min, collect supernatant in the 1.5ml centrifuge tube of no RNA enzyme；
(5) it is added into centrifuge tube and stands 10min on ice with the isometric isopropanol of supernatant, mixing；
(6) 4 DEG C of 12000g/min are centrifuged 10min, abandon supernatant；
(7) 1ml water-reducible 75% ethyl alcohol of DEPC is added, 7500g/min is centrifuged 2min；
(8) supernatant, dry 5min are abandoned；
(9) add 20ul DEPC water, dissolution；
(10) RNA concentration is measured using Nanodrop 2000, retains sample of the OD260/OD280 value between 1.8-2.0.
3. a kind of method for detecting miR5100, CXCL-12 and IL-8 relative expression quantity as described in claim 1, feature exist
In: the RNA reverse transcription reaction is PrimeScript RT reagent Kit with gDNA Eraser using kit,
The following steps are included:
(1) 5x gDNA Eraser Buffer 2.0ul, gDNA the removal reaction of genomic DNA: are added in micro tube
Eraser 1.0ul, Total RNA 1.0ug and RNase Free dH2O supplies 10ul, 42 DEG C of removal genomic DNA reactions
2min；
(2) reverse transcription reaction: RNase Free dH is separately added into the micro tube of step (1)2O 4ul、5x
PrimeScript Buffer (for Real Time) 4.0ul mixing after add RT Primer Mix1.0ul and
PrimeScript RT Enzyme Mix I 1.0ul is mixed gently；Reaction condition are as follows: 37 DEG C of reverse transcription 15min, 85 DEG C of denaturation
5s, 4 DEG C stored refrigerated.
4. a kind of method for detecting miR5100, CXCL-12 and IL-8 relative expression quantity as claimed in claim 3, feature exist
In: the RNA reverse transcription reaction condition is 37 DEG C of reverse transcription 30min.
5. a kind of method for detecting miR5100, CXCL-12 and IL-8 relative expression quantity as described in claim 1, feature exist
In: the real-time fluorescence quantitative PCR, including real-time fluorescence quantitative PCR is carried out to miR5100, real-time fluorescence is carried out to CXCL-12
Quantitative PCR and to IL-8 carry out real-time fluorescence quantitative PCR.
6. a kind of method for detecting miR5100, CXCL-12 and IL-8 relative expression quantity as claimed in claim 5, feature exist
In: described to carry out real-time fluorescence quantitative PCR to miR5100, used kit is Hieff qPCR SYBR Green Master
Mix (No Rox), qPCR reaction system are 20ul:2X SYBR Green Mix 10ul, cDNA 2ul, U6 upstream primer
0.8ul, U6 downstream primer 0.8ul, ddH2O supplies 20ul；QPCR response procedures are as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation
10s, 60 DEG C of annealing 20s, 72 DEG C of extension 20s, totally 40 recycle；95 DEG C of denaturation 15s of solubility curve, 60 DEG C of renaturation 60s, 95 DEG C of changes
Property 15s, U6 is as miR5100 internal reference.
7. a kind of method for detecting miR5100, CXCL-12 and IL-8 relative expression quantity as claimed in claim 5, feature exist
In: described to carry out real-time fluorescence quantitative PCR to CXCL-12, used kit is Hieff qPCR SYBR Green Master
Mix (No Rox), qPCR reaction system are 20ul:2X SYBR Green Mix 10ul, cDNA 2ul, GAPDH upstream primer
0.8ul, GAPDH downstream primer 0.8ul, ddH2O supplies 20ul；QPCR response procedures are as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation
10s, 60 DEG C of annealing 20s, 72 DEG C of extension 20s, totally 40 recycle；95 DEG C of denaturation 15s of solubility curve, 60 DEG C of renaturation 60s, 95 DEG C of changes
Property 15s, GAPDH is as CXCL-12 internal reference.
8. a kind of method for detecting miR5100, CXCL-12 and IL-8 relative expression quantity as claimed in claim 5, feature exist
In: described to carry out real-time fluorescence quantitative PCR to IL-8, used kit is Hieff qPCR SYBR Green Master Mix
(No Rox), qPCR reaction system are 20ul:2X SYBR Green Mix10ul, cDNA 2ul, GAPDH upstream primer
0.8ul, GAPDH downstream primer 0.8ul, ddH2O supplies 20ul；QPCR response procedures are as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation
10s, 60 DEG C of annealing 20s, 72 DEG C of extension 20s, totally 40 recycle；95 DEG C of denaturation 15s of solubility curve, 60 DEG C of renaturation 60s, 95 DEG C of changes
Property 15s, GAPDH is as IL-8 internal reference.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101415837A (en) * | 2003-07-18 | 2009-04-22 | 优基谱 | Biomarker panel for colorectal cancer |
| EP2704745A1 (en) * | 2011-05-05 | 2014-03-12 | Duke University | Methods of developing a prognosis for pancreatic cancer and predicting responsiveness to cancer therapeutics |
| CN108588218A (en) * | 2018-04-02 | 2018-09-28 | 上海交通大学医学院附属第九人民医院 | A kind of minimally invasive detection kit of serum miRNA combination |
-
2018
- 2018-11-13 CN CN201811348833.1A patent/CN109234365A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101415837A (en) * | 2003-07-18 | 2009-04-22 | 优基谱 | Biomarker panel for colorectal cancer |
| EP2704745A1 (en) * | 2011-05-05 | 2014-03-12 | Duke University | Methods of developing a prognosis for pancreatic cancer and predicting responsiveness to cancer therapeutics |
| CN108588218A (en) * | 2018-04-02 | 2018-09-28 | 上海交通大学医学院附属第九人民医院 | A kind of minimally invasive detection kit of serum miRNA combination |
Non-Patent Citations (2)
| Title |
|---|
| 于红静: "趋化因子CCL18、CXCL1促进卵巢癌增殖和转移分子机制初探", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
| 宝生物工程（大连）有限公司: "RNAiso Plus Total RNA提取试剂说明书", 《RNAISO PLUS TOTAL RNA提取试剂说明书》 * |
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