CN108220429A - Detect the primer pair of people FZD7 gene expression amounts and relative expression quantity - Google Patents
Detect the primer pair of people FZD7 gene expression amounts and relative expression quantity Download PDFInfo
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Abstract
The invention discloses the primer pairs of detection people FZD7 gene expression amounts and relative expression quantity.The primer pair of detection people FZD7 gene expression amounts and relative expression quantity disclosed by the invention, the primer pair being made of the single stranded DNA shown in sequence in sequence table 1 and the single stranded DNA shown in sequence 2 for being entitled FZD7 P.It is demonstrated experimentally that the primer pair of the present invention can detect expression contents of the FZD7 in colon cancer tissue, operation is relatively easy, as a result accurately.
Description
Technical field
The present invention relates to the primer pairs that people FZD7 gene expression amounts and relative expression quantity are detected in biotechnology.
Background technology
Colon cancer is the common malignant tumor of digestive tract for betiding colon site, is apt to occur in rectum and has a common boundary with sigmoid colon
Place, with the ratio between 40~50 years old age group incidence highest, men and women for 2~3：1.Incidence accounts for the 3rd of gastroenteric tumor.According to system
Meter, 2002, the incidence of China's colon cancer was only 7%, and by 2015, incidence turned over closely, its morbidity in recent years has
The trend of rejuvenation.The general stool blood of detection method (FOBT) experiment, glycosyl antigens c A199, gastrointestinal tract CT inspection clinically
Look into, fibergastroscopy, sigmoidoscopy etc..Therefore, it is most important to find new diagnosis of colon cancer marker.
FZD7 (frizzled class receptor 7) is a kind of acceptor molecule.Some researches show that the gene and WNT are believed
Number access is related, can be migrated with regulating cell, the cell cycle, the behaviors such as Apoptosis.In colon cancer, with cancer beside organism's phase
Than the expression of FZD7 is significantly raised.Therefore, FZD7 promises to be potential diagnosis of colon cancer marker.
Invention content
The technical problems to be solved by the invention are how to detect people FZD7 gene expression amounts or relative expression quantity.
In order to solve the above technical problems, present invention firstly provides detection or auxiliary detection people FZD7 gene expression amounts or phases
To the primer pair of expression quantity.
Detection provided by the present invention or the primer pair of auxiliary detection people FZD7 gene expression amounts or relative expression quantity, run after fame
The primer pair being made of the single stranded DNA shown in sequence in sequence table 1 and the single stranded DNA shown in sequence 2 of referred to as FZD7-P.
In order to solve the above technical problems, the present invention also provides detection or auxiliary detection mankind FZD7 gene expression amounts or phases
To the reagent set of expression quantity.
Detection provided by the present invention or the reagent set of auxiliary detection mankind FZD7 gene expression amounts or relative expression quantity,
It is made of the FZD7-P with the primer pair combined with the GAPDH gene specifics of people of entitled GAPDH-P.
In above-mentioned reagent set, the GAPDH-P is as the single stranded DNA shown in sequence in sequence table 3 and the list shown in sequence 4
Chain DNA forms.
In order to solve the above technical problems, the present invention also provides detection or auxiliary detection people FZD7 gene expression amounts or relatively
The reagent or kit of expression quantity.
Detection provided by the present invention or the reagent or reagent of auxiliary detection people FZD7 gene expression amounts or relative expression quantity
Box is made of the FZD7-P or described reagent sets with X1, and the X1 is carries out reagent needed for quantitative pcr amplification.
In mentioned reagent or kit, reagent needed for the progress quantitative pcr amplification can be qPCR MIX.QPCR MIX can
For Beijing full formula gold biotechnology (TransGen Biotech) Co., Ltd's product.
In mentioned reagent or kit, the quantitative pcr amplification can be that real-time fluorescence quantitative PCR expands.
In order to solve the above technical problems, the present invention also provides detection or auxiliary detection people FZD7 gene expression amounts or relatively
The system of expression quantity.
Detection provided by the present invention or the system of auxiliary detection people FZD7 gene expression amounts or relative expression quantity, by described
FZD7-P, the reagent set or the reagent or kit and Y1 composition, the Y 1 be carry out the required reagent of RNA extractions,
It carries out the reagent needed for reverse transcription and/or carries out instrument needed for quantitative pcr amplification.
In above system, the reagent carried out needed for RNA extractions can be TRIzol, chloroform, isopropanol and/or ethyl alcohol.
TRIzol can be Shanghai Jierui Biology Engineering Co., Ltd's product.
Reagent needed for the carry out reverse transcription can be the reagent in ReverTranscript qPCR RT Kit.
ReverTranscript qPCR RT Kit are Takara Products.
Instrument needed for the carry out quantitative pcr amplification can be real time fluorescent quantitative pcr instrument (such as ABI7300 or ABI7500
(Applied Biosystems companies of the U.S.)).
In order to solve the above technical problems, the present invention also provides identifications or auxiliary identification tumor tissues and/or cell to be
System.
Identification provided by the present invention or the system of auxiliary identification tumor tissues and/or cell, including the FZD7-P, institute
State reagent set, the reagent or kit or the detection or auxiliary detection people FZD7 gene expression amounts or relative expression quantity
System and gene quantification data processing system；The gene quantification data processing system will come from tissue to be identified for calculating
And/or the expression quantity or relative expression quantity of the FZD7 genes of cell, according to the tissue to be identified and/or the FZD7 genes of cell
Expression quantity or relative expression quantity determine it is to be identified tissue and/or cell whether be tumor tissues and/or cell.
In order to solve the above technical problems, the present invention also provides the expression quantity of detection FZD7 genes or the sides of relative expression quantity
Method.
The method of the expression quantity or relative expression quantity of detection FZD7 genes provided by the present invention, including waiting to reflect in vitro
The cDNA or RNA for determining tissue and/or cell are template, carry out PCR amplification with the FZD7-P, the annealing in the PCR amplification
Condition can be 58 DEG C of 35sec.Reaction condition is concretely：95 DEG C of pre-degeneration 1min；95 DEG C of 15s, 58 DEG C of 35sec, 40 are followed
Ring.
The reaction system during PCR amplification can be 25 μ L reaction systems, specific as follows：2 μ L cDNA solution, 12.5 μ L
QPCRMix (2 ×), FZD7-F (FZD7-F in the reaction system a concentration of 0.2 μM), (FZD7-R is in the reaction by FZD7-R
A concentration of 0.2 μM in system), it is mended with sterile distilled water to 25 μ L.
In order to solve the above technical problems, the present invention also provides identification or the sides of auxiliary identification tumor tissues and/or cell
Method.
Identification provided by the present invention or the method for auxiliary identification tumor tissues and/or cell, including：Detection comes from and waits to reflect
Determine the expression quantity or relative expression quantity of the FZD7 genes of tissue and/or cell；If the tissue to be identified and/or cell
The expression quantity or relative expression quantity of FZD7 genes are higher, it is described it is to be identified tissue and/or cell be or candidate be tumor tissues and/
Or the risk of cell is bigger, if the expression quantity or relative expression quantity of the FZD7 genes of the tissue to be identified and/or cell are got over
Low, the tissue to be identified and/or the risk that cell is or candidate is tumor tissues and/or cell are smaller.
In the above method, the tissue to be identified and/or cell can be cervical tissue and/or cervical cell.
In order to solve the above technical problems, the present invention also provides the FZD7-P, the reagent set, the reagent or examinations
Agent box or the system are following 1) -5) in it is any in application：
1) detect or assist detection people FZD7 gene expression amounts or relative expression quantity；
2) detection or auxiliary detection people FZD7 gene expression amounts or relative expression quantity product are prepared；
3) identify or assist identification tumor tissues and/or cell；
4) identification or auxiliary identification tumor tissues and/or cell products are prepared.
In above application, the tumor tissues and/or cell can be colon cancer tissue and/or cell.
It is demonstrated experimentally that the primer pair of the present invention can detect expression contents of the FZD7 in colon cancer tissue, operation is opposite
Simply, as a result accurately.
The present invention in view of in the prior art detect colon cancer sample marker deficiency, the present invention devise detection internal reference/
Target gene primer detects people's fusion relative expression quantity with fluorescent quantitative PCR technique.By adjusting drawing for two genes
Object concentration and probe concentration and ratio optimize the reaction system and reaction condition of PCR, develop one kind for detecting FZD7 genes with respect to table
Up to the kit of amount.
Using the kit of the present invention, real-time fluorescence PCR technology is detected the expression of FZD7 genes, detects
Precision is high, and easy to operate, can reduce testing cost, saves detection time.Using double calibration curve methods, by building FZD7
With the standard quantitative curve of reference gene GAPDH, the reference gene copy number of accurate quantification sample and FZD7 copy numbers, compared to
Previous ImmunohistochemistryMethods Methods, which has many advantages, such as precision height, as a result convenient for interpretation.In addition the kit is by reactant
Primer, probe needed for system carry out rational proportion and optimization, experiment condition are made to reach best, so as to which the condition for eliminating cumbersome is touched
Grommet section, greatly improves conventional efficient.The kit is specific good after tested, and high sensitivity is easy to operate.Contribute to clinic
The complementary detection of sample.
Description of the drawings
Fig. 1 is FZD7 melting curves.
Fig. 2 is GAPDH melting curves.
Fig. 3 is the amplification curve of FZD7 and GAPDH.Wherein.Left side is GAPDH amplification curves, and right side is bent for FZD7 amplifications
Line.
Fig. 4 is the relative expression quantity of 6 clinical colorectal cancer patients FZD7.
Specific embodiment
The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1
The kit for being used to detect FZD7 gene relative expression quantities of the present invention, including：
TRIzol (Shanghai Jierui Biology Engineering Co., Ltd)；
Chloroform；
Absolute ethyl alcohol；
ReverTranscript qPCR RT Kit (Takara companies)；
Detection architecture PCR reaction solution：QPCR Mix (2 ×) (the full formula gold biotechnologys in Beijing (TransGen Biotech)
Co., Ltd), each 0.2 μM of FZD7-F/FZD7-R
Wherein, primer sequence is：
FZD7-F：CATTACGGTCTCTCCTCCCC (sequence 1)
FZD7-R：AAAACACCCGTCTTTCTGCC (sequence 2)
GAPDH-F：CATGAGAAGTATGACAACAGCCT (sequence 3)
GAPDH-R：AGTCCTTCCACGATACCAAAGT (sequence 4)
Embodiment 2
The application method of kit of the present invention：
(1) the tissue RNA of extracting：Suitable flesh tissue is taken, 1mL trizol is added in and uniformly, adds in homogenate wherein grinding
Enter 0.2ml chloroforms, concussion is uniform；4 DEG C of centrifugation 10min of 14000rpm, draw supernatant layer and are transferred in another new centrifuge tube；
Isometric isopropanol is added in, abundant mixing, is stored at room temperature 10min up and down；14000rpm4 DEG C of centrifugation 10min, abandons supernatant, adds
Enter 75% ethyl alcohol 1ml, gently turn upside down washing tube wall；4 DEG C of centrifugation 5min of 14000rpm, abandon ethyl alcohol；Drying at room temperature 10-
15min adds in 20ulRNase-free water dissolutions precipitation.
(2) with reference to the ReverTranscript qPCR RT Kit kit specifications of Takara companies, RNA is inverted
For cDNA, cDNA solution is obtained, a concentration of 50 μ L/ μ L of cDNA in cDNA solution.
(3) reagent is configured：Each reaction system is 25 μ L reaction systems, specific as follows：2 μ L cDNA solution, 12.5 μ L
QPCR Mix (2 ×), FZD7-F or GAPDH-F (FZD7-F or GAPDH-F in the reaction system a concentration of 0.2 μM),
FZD7-R or GAPDH-R (FZD7-R or GAPDH-R in the reaction system a concentration of 0.2 μM), with sterile distilled water mend to
25μL.By the use of physiological saline as blank control.
(4) it detects：Detection carries out on real-time fluorescence PCR instrument, can include ABI7300, the ABI7500 (U.S. with instrument
Applied Biosystems companies) etc..Reaction condition：95 DEG C of pre-degeneration 1min；95 DEG C of 15s, 58 DEG C of 35sec, 40 cycles,
Fluorescence signal acquires when 58 DEG C of 35sec.
(5) result judges：Threshold line is adjusted to more than background signal and negative amplification line, system according to standard curve and
CT values calculate copy number automatically.
The solubility curve and amplification curve detected using the kit is shown in Fig. 1, Fig. 2 and Fig. 3.
Embodiment 3
Clinical samples are detected using kit for detecting nucleic acid of the present invention
Detect object：Clinical colorectal cancer patients 6, (all samples are organized for the colon cancer I phases).The colon cancer of each patient
It is organized by tissue and colon cancer to 6 pairs altogether of detection, sample to be tested is detected by the application method of 2 kit of embodiment.Each
3 repetitions of sample, 1 part of blank control.Detection time only needs 100 minutes.
The results are shown in table below for 6 clinical colorectal cancer patients pattern detections：
Fig. 4 is shown in 6 clinical colorectal cancer patients pattern detection result summaries.
For Fig. 4 the results show that can detect expression contents of the FZD7 in colon cancer tissue using the primer pair, operation is opposite
Simply, as a result accurately.
The present invention in view of in the prior art detect colon cancer sample marker deficiency, the present invention devise detection internal reference/
Target gene primer detects people's fusion relative expression quantity with fluorescent quantitative PCR technique.By adjusting drawing for two genes
Object concentration and probe concentration and ratio optimize the reaction system and reaction condition of PCR, develop one kind for detecting FZD7 genes with respect to table
Up to the kit of amount.
Using the kit of the present invention, real-time fluorescence PCR technology is detected the expression of FZD7 genes, detects
Precision is high, and easy to operate, can reduce testing cost, saves detection time.Using double calibration curve methods, by building FZD7
With the standard quantitative curve of reference gene GAPDH, the reference gene copy number of accurate quantification sample and FZD7 copy numbers, compared to
Previous ImmunohistochemistryMethods Methods, which has many advantages, such as precision height, as a result convenient for interpretation.In addition the kit is by reactant
Primer, probe needed for system carry out rational proportion and optimization, experiment condition are made to reach best, so as to which the condition for eliminating cumbersome is touched
Grommet section, greatly improves conventional efficient.The kit is specific good after tested, and high sensitivity is easy to operate.Contribute to clinic
The complementary detection of sample.
Claims (10)
1. detection or the primer pair of auxiliary detection people FZD7 gene expression amounts or relative expression quantity are entitled FZD7-P by sequence
The primer pair of single stranded DNA composition shown in single stranded DNA and sequence 2 in list shown in sequence 1.
2. detection or the reagent set of auxiliary detection mankind FZD7 gene expression amounts or relative expression quantity, as described in claim 1
FZD7-P is formed with the primer pair combined with the GAPDH gene specifics of people of entitled GAPDH-P.
3. reagent set according to claim 2, it is characterised in that：The GAPDH-P is as shown in sequence in sequence table 3
Single stranded DNA composition shown in single stranded DNA and sequence 4.
4. detection or the reagent or kit of auxiliary detection people FZD7 gene expression amounts or relative expression quantity, by claim 1 institute
It states reagent set described in primer pair or Claims 2 or 3 to form with X1, the X1 is carries out reagent needed for quantitative pcr amplification.
5. reagent according to claim 4 or kit, it is characterised in that：Reagent needed for the carry out quantitative pcr amplification
For qPCR MIX.
6. detection or auxiliary detection people FZD7 gene expression amounts or relative expression quantity system, as described in claim 1 primer pair,
Reagent set described in Claims 2 or 3 or the reagent of claim 4 or 5 or kit are formed with Y1, and the Y1 is carries out
The required reagent of RNA extractions carries out the reagent needed for reverse transcription and/or carries out instrument needed for quantitative pcr amplification.
7. identification or the system of auxiliary identification tumor tissues and/or cell, including primer pair, claim 2 described in claim 1
Or 3 reagent sets, the reagent of claim 4 or 5 or kit or system and gene quantification number described in claim 6
According to processing system；The gene quantification data processing system is used to calculate the FZD7 bases from tissue to be identified and/or cell
The expression quantity or relative expression quantity of cause, according to the expression quantity or opposite table of the tissue to be identified and/or the FZD7 genes of cell
Determine whether tissue and/or cell to be identified are tumor tissues and/or cell up to amount.
8. following methods 1) or 2)：
1) method of the expression quantity or relative expression quantity of detection FZD7 genes, including in vitro tissue and/or cell to be identified
CDNA or RNA be template, the primer pair described in claim 1 carries out PCR amplification, and the annealing conditions in the PCR amplification are
58℃35sec。
2) method identified or assist identification tumor tissues and/or cell, including：Detection is from tissue to be identified and/or cell
FZD7 genes expression quantity or relative expression quantity；If the expression quantity of the FZD7 genes of the tissue to be identified and/or cell
Or relative expression quantity is higher, the tissue to be identified and/or the risk that cell is or candidate is tumor tissues and/or cell are got over
Greatly, if the expression quantity or relative expression quantity of the FZD7 genes of the tissue to be identified and/or cell are lower, described to be identified group
It knits and/or cell is or candidate is tumor tissues and/or cell risk is smaller.
9. primer pair described in claim 1, reagent set, the reagent of claim 4 or 5 or reagent described in Claims 2 or 3
Box or the system of claim 6 or 7 are following 1) -5) in it is any in application：
1) detect or assist detection people FZD7 gene expression amounts or relative expression quantity；
2) detection or auxiliary detection people FZD7 gene expression amounts or relative expression quantity product are prepared；
3) identify or assist identification tumor tissues and/or cell；
4) identification or auxiliary identification tumor tissues and/or cell products are prepared.
10. it is applied described in system, claim 8 the method or claim 9 according to claim 7, it is characterised in that：
Described in claim 7 or 8 it is to be identified tissue and/or cell be uterine neck cervical tissue and/or uterine neck cervical cell；Claim
Tumor tissues described in 9 and/or cell are colon cancer tissue and/or cell.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010037041A2 (en) * | 2008-09-26 | 2010-04-01 | Oncomed Pharmaceuticals, Inc. | Frizzled-binding agents and uses thereof |
| CN105506131A (en) * | 2016-01-15 | 2016-04-20 | 苏州吉诺瑞生物科技有限公司 | Primer pair for detecting people AEG-1 gene expression quantity and relative expression quantity |
-
2016
- 2016-12-14 CN CN201611151157.XA patent/CN108220429A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010037041A2 (en) * | 2008-09-26 | 2010-04-01 | Oncomed Pharmaceuticals, Inc. | Frizzled-binding agents and uses thereof |
| CN105506131A (en) * | 2016-01-15 | 2016-04-20 | 苏州吉诺瑞生物科技有限公司 | Primer pair for detecting people AEG-1 gene expression quantity and relative expression quantity |
Non-Patent Citations (2)
| Title |
|---|
| BOYA DENG,ET AL.: "Down-regulation of Frizzled-7 expression inhibits migration, invasion, and epithelial–mesenchymal transition of cervical cancer cell lines", 《MEDICAL ONCOLOGY》 * |
| KOJI UENO,ET AL.: "Frizzled-7 as a Potential Therapeutic Target in Colorectal Cancer", 《NEOPLASIA》 * |
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