CN101948912A - Human transient receptor potential channel protein fluorescence quantitative PCR detection kit and preparation method and application thereof - Google Patents
Human transient receptor potential channel protein fluorescence quantitative PCR detection kit and preparation method and application thereof Download PDFInfo
- Publication number
- CN101948912A CN101948912A CN2010101988157A CN201010198815A CN101948912A CN 101948912 A CN101948912 A CN 101948912A CN 2010101988157 A CN2010101988157 A CN 2010101988157A CN 201010198815 A CN201010198815 A CN 201010198815A CN 101948912 A CN101948912 A CN 101948912A
- Authority
- CN
- China
- Prior art keywords
- gene
- trpc1
- trpc7
- sptting plate
- sequence
- 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
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a human transient receptor potential channel (TRPC) protein fluorescence quantitative PCR detection kit and a preparation method and application thereof. The detection kit comprises a box body, wherein reaction plates (1) and a positive reference substance (2) are arranged in the box body. The detection kit is characterized in that a plurality of reaction holes are arranged in the reaction plates (1); the reaction plates (1) contain a fluorescence quantitative PCR standard curve reaction plate (11) and a fluorescence quantitative PCR sample under test reaction plate (12); the target gene TRPC1-TRPC7 reaction mixed liquid and the reference gene IP08 reaction mixed liquid are placed in the fluorescence quantitative PCR standard curve reaction plate (11) and the fluorescence quantitative PCR sample under test reaction plate (12); and the target gene TRPC1-TRPC7 reaction mixed liquid and the reference gene IP08 reaction mixed liquid are placed in the reaction holes. The detection kit of the invention is characterized by convenient preparation and storage and good operability and repeatability, various detection kits can be designed according to different TRPC gene research emphases, and the TRPC relatively quantitative analysis of a plurality of samples can be completed at once.
Description
Technical field
The present invention relates to people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit and its production and use.Belong to biological technical field.
Background technology
Transient receptor potential channel (is called for short: TRPC) be that a kind of non-selective cationic channel family (comprises TRPC1～TRPC7), calcium ion had permeability, this family protein has six high conservatives and strides the film district, can be divided into 4 subgroup: TRPC1 according to the similarity of 26S Proteasome Structure and Function; TRPC2; TRPC3, TRPC6 and TRPC7; TRPC4 and TRPC5.TRPC can constitute functional SOCC passage by homology or allos tetramer mode, the latter mediates generation SOCE and causes stream in the cell calcium, the TRPC abnormal expression causes the variable effect cell activities of SOCE, participate in multiple good malignant disease, for example: respiratory system disease: asthma, chronic obstructive pulmonary disease, idiopathic pulmonary hypertension; Cardiovascular system diseases: myocardial hypertrophy, hypertension, vasculitis; Urinary system: FSG; Nervous system disorders: neurodegeneration, muscular dystrophy; Disease of immune system: T/B cell immune response (inflammatory reaction) and tumor disease: liver cancer, prostate cancer, mammary cancer, ovarian cancer, cancer of the stomach and the esophageal carcinoma etc.Especially the effect of TRPC genetic expression in tumor development begins to be subjected to extensive concern.Therefore, the molecule mechanism of illustrating TRPC is for the diagnosis of various diseases with treat most important.
Real-time fluorescence quantitative polymerase chain reaction technology (RT-qPCR) is meant in the PCR reaction system and adds fluorophor, real-time collecting accumulation fluorescence signal intensity is monitored whole PCR process, set up real-time amplification curve and determine Ct value, the method for by typical curve unknown template being carried out the initial point quantitative analysis at last.Have highly sensitive, high specificity, good, the simple operation and other advantages of linear relationship, can high-throughoutly carry out that gene quantification detects and the relative expression analyzes.The aspect such as gene rapid detection, early diagnosis, gene type that is widely used in human various diseases.Adopting this method to carry out the gene quantification analysis at present at first needs design, the specific primer of synthetic gene, and repeatedly this primer is carried out efficient and specific detection, and finishing screen is selected suitable primer.Therefore, process is loaded down with trivial details relatively, consuming time, operability is low.
Summary of the invention
First purpose of the present invention is for a kind of people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit is provided.
Second purpose of the present invention is for a kind of preparation method of people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit is provided.
The 3rd purpose of the present invention is for a kind of purposes of people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit is provided.
First purpose of the present invention can reach by following measure:
People's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit, it comprises box body, in box body, be provided with Sptting plate and positive reference substance, it is characterized in that: be provided with some reacting holes in Sptting plate, described Sptting plate comprises quantitative fluorescent PCR typical curve Sptting plate and quantitative fluorescent PCR testing sample Sptting plate; In quantitative fluorescent PCR typical curve Sptting plate, quantitative fluorescent PCR testing sample Sptting plate, be provided with goal gene TRPC1～TRPC7 reaction mixture and internal control gene IP08 reaction mixture, described goal gene TRPC1～TRPC7 reaction mixture comprises TRPC1～TRPC7 gene primer sequence, SYBR fluorescence dye, archaeal dna polymerase and reactive ion damping fluid, and described internal control gene IP08 reaction mixture comprises IP08 gene primer sequence, SYBR fluorescence dye, archaeal dna polymerase and reactive ion damping fluid; Described goal gene TRPC1～TRPC7 reaction mixture and internal control gene IP08 reaction mixture are arranged in the reacting hole.
The present invention is with transient receptor potential channel (TRPC) gene transcript (messenger RNA(mRNA) among the Gene Bank, mRNA) be template, design, synthetic people TRPC gene primer, by stdn real-time fluorescence quantitative polymerase chain reaction technology, filter out efficient, special TRPC gene primer, and transient receptor potential channel (TRPC) the gene by fluorescence quantitative detection kit that these primers are made into other PCR reacted constituent.
First purpose of the present invention can also reach by following measure:
The further embodiment of first purpose of the present invention is it is characterized in that:
1) nucleotide sequence of described TRPC1 gene primer sequence is as sequence table SEQ ID №: shown in 1;
2) nucleotide sequence of described TRPC2 gene primer sequence is as sequence table SEQ ID №: shown in 2;
3) nucleotide sequence of described TRPC3 gene primer sequence is as sequence table SEQ ID №: shown in 3;
4) nucleotide sequence of described TRPC4 gene primer sequence is as sequence table SEQ ID №: shown in 4;
5) nucleotide sequence of described TRPC5 gene primer sequence is as sequence table SEQ ID №: shown in 5;
6) nucleotide sequence of described TRPC6 gene primer sequence is as sequence table SEQ ID №: shown in 6;
7) nucleotide sequence of described TRPC7 gene primer sequence is as sequence table SEQ ID №: shown in 7;
8) nucleotide sequence of described IP08 gene primer sequence is as sequence table SEQ ID №: shown in 8.
The further embodiment of first purpose of the present invention is characterized in that: positive reference substance is human brain tissue cDNA.
The further embodiment of first purpose of the present invention is characterized in that: described quantitative fluorescent PCR typical curve Sptting plate, quantitative fluorescent PCR testing sample Sptting plate respectively are provided with 96 reacting holes, constitute 96 orifice plates.
Second purpose of the present invention can reach by taking following technical scheme:
The preparation method of people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit is characterized in that comprising the steps:
1) prepares box body and be placed in box intravital quantitative fluorescent PCR typical curve Sptting plate and quantitative fluorescent PCR testing sample Sptting plate, the reacting hole of goal gene TRPC1～TRPC7 and internal control gene IP08 is set by the zone in described quantitative fluorescent PCR typical curve Sptting plate and quantitative fluorescent PCR testing sample Sptting plate, and each sample is provided with a repeating hole; Each gene is provided with two no template negative control-NTC;
2) purpose of design gene TRPC1～TRPC7 and internal control gene IP08 primer are measured the efficient and the specificity of described primer;
3) institute is surveyed primer and mix with SYBR fluorescence dye, archaeal dna polymerase and reactive ion damping fluid respectively, constitute goal gene TRPC1～TRPC7 reaction mixture and internal control gene IP08 reaction mixture respectively;
4) goal gene TRPC1～TRPC7 reaction mixture and internal control gene IP08 reaction mixture are added on respectively in aforementioned corresponding reacting hole, constitute goal gene TRPC1～TRPC7 and each Sptting plate of internal control gene IP08 quantitative fluorescent PCR;
5) preparation human brain tissue cDNA is .TRPC1～TRPC7 gene masculine control sample, gets during detection to be added in the reacting hole of typical curve Sptting plate after this sample becomes 5 concentration gradients by 2 times of doubling dilutions, constitutes the gradient reacting hole.
The 3rd purpose of the present invention can reach by taking following technical scheme:
The purposes of people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit is characterized in that: can be used for the analysis and research of the good malignant disease of human TRPC gene-correlation.
The invention has the beneficial effects as follows:
The invention provides a kind of people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit, it is convenient that this test kit is made, preserved, operability and good reproducibility, can carry out the variation design according to the difference of TRPC gene studies emphasis, can disposablely finish the TRPC relative quantitative assay of polygene multiple sample.Summarize following beneficial effect:
1, efficient, special: as to contain special TRPC1～TRPC7 gene primer in the mixed solution of test kit Sptting plate, can carry out the analysis of TRPC gene quantification efficiently.Solved the difficult problem of investigator design, synthetic, screening TRPC gene primer.
2, quantitatively accurately and reliably: the actual primer efficient of each gene can detect the miscalculation of having avoided actual efficiency and having brought with reference to difference between the efficient by human brain tissue cDNA (positive control) in the test kit.
3, easy to operate: the investigator in use, the cDNA sample to be measured that only need add recommended amounts just can disposablely be finished all T RPC quantitative analyses, has simplified the preparation of PCR reaction system.
4, modular design: the making of Sptting plate (sample detection quantity, focus TRPC gene) can require to change according to research.
5, make simply, strong operability, preservation, convenient transportation, applied widely.
Description of drawings
Fig. 1 a is the structural representation of the positive reference substance of test kit of the present invention.
Fig. 1 b is the structural representation of the Sptting plate of test kit of the present invention.
Fig. 1 c is the structural representation of the quantitative fluorescent PCR typical curve Sptting plate of test kit of the present invention.
Fig. 1 d is the structural representation of the quantitative fluorescent PCR testing sample Sptting plate of test kit of the present invention.
Wherein,
Among Fig. 1 c :-1 ,-2 ,-3 ,-4 ,-5 expressions: TRPC and IP08 gene primer typical curve 5 gradient proportional diluted holes; NTC represents: no template negative control.A, b, c, d, e, f, g and h correspond to respectively: TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6, TRPC7 and IP08.
Among Fig. 1 d: s1, s2, s3, s4, s5 represents: testing sample; NTC represents: no template negative control.A, b, c, d, e, f, g and h correspond to respectively: TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6, TRPC7 and IP08.
Fig. 2 a is an internal control gene IP08 quantitative fluorescent PCR canonical plotting of the present invention.
Fig. 2 b is an internal control gene IP08 fluorescent quantitative PCR graphic representation of the present invention.
Fig. 2 c is internal control gene IP08 quantitative fluorescent PCR product melt curve analysis figure of the present invention
Fig. 2 d is internal control gene IP08 quantitative fluorescent PCR product sequencing figure of the present invention.
Fig. 2 e is internal control gene IP08 quantitative fluorescent PCR agarose gel electrophoresis figure of the present invention.
Fig. 3 a is the object of the invention gene TRPC1 quantitative fluorescent PCR canonical plotting.
Fig. 3 b is the object of the invention gene TRPC1 fluorescent quantitative PCR graphic representation.
Fig. 3 c is the object of the invention gene TRPC1 quantitative fluorescent PCR product melt curve analysis figure.
Fig. 3 d is the object of the invention gene TRPC1 quantitative fluorescent PCR product sequencing figure.
Fig. 3 e is the object of the invention gene TRPC1 quantitative fluorescent PCR agarose gel electrophoresis figure.
Fig. 4 a is the object of the invention gene TRPC2 quantitative fluorescent PCR canonical plotting.
Fig. 4 b is the object of the invention gene TRPC2 fluorescent quantitative PCR graphic representation.
Fig. 4 c is the object of the invention gene TRPC2 quantitative fluorescent PCR product melt curve analysis figure.
Fig. 4 d is the object of the invention gene TRPC2 quantitative fluorescent PCR product sequencing figure.
Fig. 4 e is the object of the invention gene TRPC2 quantitative fluorescent PCR agarose gel electrophoresis figure.
Fig. 5 a is the object of the invention gene TRPC3 quantitative fluorescent PCR canonical plotting.
Fig. 5 b is the object of the invention gene TRPC3 fluorescent quantitative PCR graphic representation.
Fig. 5 c is the object of the invention gene TRPC3 quantitative fluorescent PCR product melt curve analysis figure.
Fig. 5 d is the object of the invention gene TRPC3 quantitative fluorescent PCR product sequencing figure.
Fig. 5 e is the object of the invention gene TRPC3 quantitative fluorescent PCR agarose gel electrophoresis figure.
Fig. 6 a is the object of the invention gene TRPC4 quantitative fluorescent PCR canonical plotting.
Fig. 6 b is the object of the invention gene TRPC4 fluorescent quantitative PCR graphic representation.
Fig. 6 c is the object of the invention gene TRPC4 quantitative fluorescent PCR product melt curve analysis figure.
Fig. 6 d is the object of the invention gene TRPC4 quantitative fluorescent PCR product sequencing figure.
Fig. 6 e is the object of the invention gene TRPC4 quantitative fluorescent PCR agarose gel electrophoresis figure.
Fig. 7 a is the object of the invention gene TRPC5 quantitative fluorescent PCR canonical plotting.
Fig. 7 b is the object of the invention gene TRPC5 fluorescent quantitative PCR graphic representation.
Fig. 7 c is the object of the invention gene TRPC5 quantitative fluorescent PCR product melt curve analysis figure.
Fig. 7 d is the object of the invention gene TRPC5 quantitative fluorescent PCR product sequencing figure.
Fig. 7 e is the object of the invention gene TRPC5 quantitative fluorescent PCR agarose gel electrophoresis figure.
Fig. 8 a is the object of the invention gene TRPC6 quantitative fluorescent PCR canonical plotting.
Fig. 8 b is the object of the invention gene TRPC6 fluorescent quantitative PCR graphic representation.
Fig. 8 c is the object of the invention gene TRPC6 quantitative fluorescent PCR product melt curve analysis figure.
Fig. 8 d is the object of the invention gene TRPC6 quantitative fluorescent PCR product sequencing figure.
Fig. 8 e is the object of the invention gene TRPC6 quantitative fluorescent PCR agarose gel electrophoresis figure.
Fig. 9 a is the object of the invention gene TRPC7 quantitative fluorescent PCR canonical plotting.
Fig. 9 b is the object of the invention gene TRPC7 fluorescent quantitative PCR graphic representation.
Fig. 9 c is the object of the invention gene TRPC7 quantitative fluorescent PCR product melt curve analysis figure.
Fig. 9 d is the object of the invention gene TRPC7 quantitative fluorescent PCR product sequencing figure.
Fig. 9 e is the object of the invention gene TRPC7 quantitative fluorescent PCR agarose gel electrophoresis figure.
Figure 10 a is the object of the invention gene TRPC1 and the expression synoptic diagram of TRPC6 in the NSCLC tissue.
Figure 10 b is the object of the invention gene TRPC2, TRPC3, TRPC4, TRPC5 and the TRPC7 expression synoptic diagram in the NSCLC tissue.
Embodiment
The present invention is described in further detail below in conjunction with specific embodiment, Application Example and accompanying drawing:
Specific embodiment:
With reference to Fig. 1 a, Fig. 1 b, Fig. 1 c and Fig. 1 d, present embodiment comprises box body, be provided with Sptting plate 1 and positive reference substance 2 in box body, be provided with six group reaction holes 3 in Sptting plate 1, Sptting plate 1 comprises quantitative fluorescent PCR typical curve Sptting plate 11 and quantitative fluorescent PCR testing sample Sptting plate 12; In quantitative fluorescent PCR typical curve Sptting plate 11, quantitative fluorescent PCR testing sample Sptting plate 12, be provided with goal gene TRPC1～TRPC7 reaction mixture and internal control gene IP08 reaction mixture, goal gene TRPC1～TRPC7 reaction mixture comprises TRPC1～TRPC7 gene primer sequence, SYBR fluorescence dye, archaeal dna polymerase and reactive ion damping fluid, and internal control gene IP08 reaction mixture comprises IP08 gene primer sequence, SYBR fluorescence dye, archaeal dna polymerase and reactive ion damping fluid; Described goal gene TRPC1～TRPC7 reaction mixture and internal control gene IP08 reaction mixture are arranged in the reacting hole 3.
In the present embodiment, described positive reference substance 2 is human brain tissue cDNA.Described quantitative fluorescent PCR typical curve Sptting plate 11, quantitative fluorescent PCR testing sample Sptting plate 12 are 96 orifice plates, each testing gene is provided with no template negative control NTC, each sample is provided with reaction repeated hole 3, can carry out the detection of TRPC1～TRPC7 and IP08 gene to 5 samples simultaneously.
The described people's transient receptor potential channel of present embodiment albumen (TRPC) fluorescent quantificationally PCR detecting kit mainly is achieved through the following technical solutions: 1, TRPC1～7 genes and internal control gene IP08 design of primers are with synthetic; 2, the preparation of primer efficient and specific assay 3, goal gene TRPC1～7 and each Sptting plate of internal control gene IP08 quantitative fluorescent PCR; 4, the preparation of TRPC1～7 gene masculines contrasts (human brain tissue cDNA).
Specific as follows:
1.TRPC1～7 genes and internal control gene IP08 design of primers are with synthetic
With reference to table 1, be the corresponding primer of stencil design with GeneBank goal gene TRPC1～TRPC7 and IP08 internal control gene transcription product (mRNA), method of design adopts the online design software of Primer3: primer send Dalian TAKARA company synthetic.
Table 1 people TRPC and IP08 primer information table
| The TRPC gene | Gene Bank sequence number | Primer sequence (left/right) | Product size (bp) |
| TRPC1 | NM_003304 | 5’-ttgtggaggtggaattcagg-3’ 5’-cgtttgtcaagaggctcgtc-3’ | 148 |
| TRPC2 | NR_002720 | 5’-tcatggtcattgtgctgctc-3’ 5’-actccacgtcagcatcatcc-3’ | 84 |
| TRPC3 | NM_003305 | 5’-cagccaacacgttatcagca-3’ 5’-cctcagttgcttggctcttg-3’ | 172 |
| TRPC4 | NM_001135958 | 5’-cgaaagggttaacctgcaaa-3’ 5’-cagggactgcagtgtctcaa-3’ | 83 |
| TRPC5 | NM_012471 | 5’-gtgctgctgaacatgctgat-3’ 5’-gcttcgtccttgcaaacttc-3’ | 94 |
| TRPC6 | NM_004621 | 5’-cagacaatggcggtcaagtt-3’ 5’-tggtccacgcattatcttcc-3’ | 117 |
| TRPC7 | NM_020389 | 5’-gttaaaaccctgccaaacga-3’ 5’-tcccagatttccttgcattc-3’ | 143 |
| IP08 | NM_006390 | 5’-aaccaaggggtggttcattc-3’ 5’-ttgccacagctcttcatcct-3’ | 120 |
2.TRPC gene primer efficient (efficiency:E) and specific assay
Adopt German Qiagen company's PCR kit for fluorescence quantitative (Quanti Tect SYBR Green PCR), make TRPC1～7 goal gene and IP08 internal control gene primer typical curve (get and detect after the capable 2 times of doubling dilutions of human brain tissue cDNA sample become 5 each concentration gradient), solubility curve with the positive contrast of human brain tissue cDNA, and to capable 1.5% agarose gel electrophoresis of pcr amplification product and order-checking to determine each gene primer efficient and specificity, finishing screen is selected efficient and special gene primer.
With reference to Fig. 2 a～Fig. 2 e, typical curve shows primer average efficiency (E value)=100%, relation conefficient (R
2)=0.996, slope=-3.332.
With reference to Fig. 3 a～Fig. 3 e, typical curve shows primer average efficiency (E value)=97%, relation conefficient (R
2)=0.88, slope=-3.395.
With reference to Fig. 4 a～Fig. 4 e, typical curve shows primer average efficiency (E value)=104.4%, relation conefficient (R
2)=0.977, slope=-3.221.
With reference to Fig. 5 a～Fig. 5 e, typical curve shows primer average efficiency (E value)=93.1%, relation conefficient (R
2)=0.99, slope=-3.500.
With reference to Fig. 6 a～Fig. 6 e, typical curve shows primer average efficiency (E value)=99.9%, relation conefficient (R
2)=0.939, slope=-3.325.
With reference to Fig. 7 a～Fig. 7 e, typical curve shows primer efficient (E value)=98.1%, relation conefficient (R
2)=0.995, slope=-3.369.
With reference to Fig. 8 a～Fig. 8 e, typical curve shows primer average efficiency (E value)=99.3%, relation conefficient (R
2)=0.946, slope=-3.339.
With reference to Fig. 9 a～Fig. 9 e, typical curve shows primer average efficiency (E value)=103.7%, relation conefficient (R
2)=0.997, slope=-3.237.
In sum, with the positive contrast of human brain tissue cDNA, make TRPC1～7 genes and IP08 gene primer quantitative fluorescent PCR typical curve, through measuring (＞5 times) repeatedly, confirm that designed primer amplification efficient is near 100%, goal gene and internal control gene primer efficient differs＜and 5%, the melt curve analysis of each gene amplification product presents simple spike; In addition, the amplified production row agarose gel electrophoresis see single band and with estimated molecular weight coincide, through order-checking confirm with the target sequence matching rate greater than 99%, show that designed primer is efficient, special, be applicable to the Quantitative Study of human TRPC gene.
3. the preparation of goal gene TRPC1～7 and internal control gene IP08 quantitative fluorescent PCR Sptting plate (is example with TRPC)
1. the preparation of gene primer working fluid: add no RNA enzyme water dissolution TRPC1～TRPC7 and IP08 gene upstream and downstream primer by primer dissolving requirement, make 100 μ M mother liquors.Get each 20 μ l of gene upstream and downstream primer mother liquor, add 160 μ l DEPC water, make this gene primer working fluid.
2. gene reaction reagent mixture formula following (lucifuge low temperature down preparation):
| 2 * SYBR Green (fluorescence dye) | 12.5 |
| TRPC upstream and downstream primer mixed solution (10uM) | 1 |
| No RNA enzyme water | 6.5 |
| Total | 20μl |
Annotate: on show the compound method that the reaction reagent mixed solution is a reaction system, can adjust according to sample size in the research.
3. the preparation of quantitative fluorescent PCR Sptting plate
(1) gets 96 orifice plates, be labeled as TRPC and IP08 gene: typical curve Sptting plate (positive control-human brain tissue cDNA) and testing sample Sptting plate.Every sample is provided with 2 repetitions.
(2) get above-mentioned each gene reaction reagent mixed solution 20 μ l, be added in the reacting hole of each Sptting plate corresponding zone, 96 hole, promptly be made into--quantitative fluorescent PCR Sptting plate (typical curve Sptting plate and testing sample Sptting plate).
(3) sealing, lucifuge, 4 ℃ of preservations
Annotate: owing to a Sptting plate can detect simultaneously to 5 each testing sample TRPC1～TRPC7 and internal control gene IP08, in order to satisfy the needs of different researchs:
A, this test kit can be made into plurality of specifications, wherein comprise the Sptting plate of different numbers, to satisfy the needs of different sample detection quantity.
B, according to the difference of TRPC research emphasis, can carry out the modularization preparation to TRPC1～TRPC7 in the Sptting plate, to satisfy needs to different TRPC gene studiess.
4, the preparation of TRPC1～TRPC7 gene masculine contrast (human brain tissue cDNA)
1., collect excision human brain tissue, liquid nitrogen preservation
2., adopt Trizol test kit (invetrogen company), by specification to require to extract the total RNA of human brain tissue.(Ambion company) removes residual DNA in total RNA sample with TURBO DNA-free reagent.
3., prepare the reverse transcription reaction system by the explanation of iScript reverse transcription test kit: the reaction system volume is 20 μ l, wherein contains RNA 1 μ g.The reverse transcription condition is: 25 ℃, and 5min; 42 ℃, 30min; 85 ℃, 5min.
4., the reverse transcription PCR product is human brain tissue cDNA positive control ,-80 ℃ of preservations.
5., when experiment, utilize human brain tissue cDNA as positive control, get stoste and carry out after 2 times of doubling dilutions become five gradients, be added on the gradient reacting hole of typical curve Sptting plate respectively, can be in order to detect TRPC and IP08 gene primer efficient and specificity.
Application Example:
Below in conjunction with application example this test kit is described in further detail:
People's transient receptor potential channel (TRPC) gene by fluorescence quantitative detection kit applicating example: TRPC expression of gene check and analysis in the non-small cell lung cancerous tissue.
1, the extraction of nonsmall-cell lung cancer (NSCLC) total tissue RNA
1., use Trizol reagent, extract total RNA by company's product description.Remove residual DNA in total RNA sample with TURBO DNA-free reagent.
2., determined by ultraviolet spectrophotometry A260nm, A280nm optical density(OD), by formula [RNA] (μ g/ml)=40 * extension rate * A260nm calculates RNA concentration; 1.2% denaturing formaldehyde agarose gel electrophoresis.
The RNA sample quality that is used for this research requires, and A260 and A280 ratio do not have disperse greater than the high-visible 28s of 1.8,1.2% denaturing formaldehyde agarose gel electrophoresis and IP08 Liang Tiao district band, banding pattern.
2, the synthetic cDNA of reverse transcription PCR
1., adopt iScript reverse transcription test kit, reaction system is 20 μ l, wherein containing total RNA amount is 1 μ g.
2., the reverse transcription condition is: 25 ℃, 5min; 42 ℃, 30min; 85 ℃, 5min.
3., product is cDNA, puts-80 ℃ of preservations.
4., when detecting, each cDNA sample adds 180 μ L does not have RNA enzyme water, makes working fluid.
3, fluorescence quantitative PCR detection NSCLC tissue T RPC mRNA expresses
1., take out Sptting plate in people's transient receptor potential channel (TRPC) gene by fluorescence quantitative detection kit, lucifuge is as on ice.
2., take out 5 μ l testing sample cDNA working fluids and add to Sptting plate TRPC gene and IP08 internal control gene sample to be tested zone; Get 1 μ l human brain tissue positive control and add to TRPC box gene IP08 internal control gene typical curve zone.
3., reaction parameter is set, instrument adopts Bio-rad I Q5 PCR instrument to carry out quantitative fluorescent PCR reaction (with reference to table 2)
4., product is put-80 ℃ of cryogenic refrigerators preservations.
Table 2TRPC gene real-time fluorescence quantitative PCR reaction parameter table
4, PCR product agarose gel electrophoresis and order-checking
(1) sepharose of preparation 1.5%; Prerunning 10min;
(2) get TRPC quantitative fluorescent PCR product 10 μ l, add 6 * Loading Buffer, 2 μ l, join behind the mixing on the sepharose in the sample hole, connect power supply, it is 120V that voltage is set, electrophoresis 30min; Carry out gel imaging system scanning then, take a picture.
(3) PCR product order-checking.
5. data processing and statistical study:
Compile each gene C t value, adopt Pfaff l formula to calculate each gene relative expression quantity (Figure 10 a, Figure 10 b):
Original RNA copy number=(E
Goal gene)
-Ct goal gene/ (E
Internal control gene)
-Ct internal control gene
With reference to Figure 10 a, Figure 10 b, utilization people's transient receptor potential channel (TRPC) gene by fluorescence quantitative detection kit detects NSCLC tissue T RPC expression of gene.
Adopt TRPC1～7 gene primers of above-mentioned checking, producer's transient receptor potential channel (TRPC) gene by fluorescence quantitative detection kit.Use this test kit to detect NSCLC tissue T RPC expression conditions: to randomly draw 6 routine NSCLC tissues and carry out TRPC fluorescent quantitation relative expression analysis, detected result finds that NSCLC expresses TRPC1,3,4 and 6mRNA, do not detect TRPC2,5 and 7mRNA express.Various kinds is originally calculated Mean Ct value respectively, adopt Pfaff l formula to calculate the goal gene relative expression quantity, analyze the expression abundance of each TRPC gene in the NSCLC tissue: TRPC1 ≈ TRPC6＞＞TRPC3＞TRPC4.The expression amount of TRPC3 and TRPC4 is about 1/8 and 1/25 of TRPC1 respectively.
Annotate: fluorescence signal intensity reached the cycle number of threshold value when the Ct value was gene amplification.
Above result shows: people's transient receptor potential channel of the present invention (TRPC) gene by fluorescence quantitative detection kit can be efficiently, efficiently unknown sample TRPC gene is carried out relative relative quantitative assay.Be expected to apply to rapid detection, Quantitative Study of human various diseases TRPC gene etc., significant.
The present invention is by the synthetic specific TRPC primer of design, selects the efficient special TRPC1～TRPC7 primer of a cover through testing sieve repeatedly, utilizes these primers, is prepared into a series of TRPC detection by quantitative Sptting plate, is used for clinical and fundamental research.
Sequence table
＜110〉The Second Affiliated Hospital of Guangzhou Medical School; Guangzhou Inst. of Respiratory Diseases
＜120〉people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit and its production and use
<160>1
<210>1
<211>
<212>DNA
＜213〉artificial sequence
<220>
＜223〉TRPC1 gene primer sequence is used to prepare people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit
<400>1
Upstream primer sequence: 5 '-ttgtggaggtggaattcagg-3 ',
Downstream primer sequence: 5 '-cgtttgtcaagaggctcgtc-3 ';
<210>2
<211>
<212>DNA
＜213〉artificial sequence
<220>
＜223〉TRPC2 gene primer sequence is used to prepare people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit
<400>2
Upstream primer sequence: 5 '-tcatggtcattgtgctgctc-3 ',
Downstream primer sequence: 5 '-actccacgtcagcatcatcc-3 ';
210>3
<211>
<212>DNA
＜213〉artificial sequence
<220>
＜223〉TRPC3 gene primer sequence is used to prepare people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit
<400>3
Upstream primer sequence: 5 '-cagccaacacgttatcagca-3 ',
Downstream primer sequence: 5 '-cctcagttgcttggctcttg-3 ';
210>4
<211>
<212>DNA
＜213〉artificial sequence
<220>
＜223〉TRPC4 gene primer sequence is used to prepare people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit
<400>4
Upstream primer sequence: 5 '-cgaaagggttaacctgcaaa-3 ',
Downstream primer sequence: 5 '-cagggactgcagtgtctcaa-3 ';
210>5
<211>
<212>DNA
＜213〉artificial sequence
<220>
＜223〉TRPC5 gene primer sequence is used to prepare people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit
<400>5
Upstream primer sequence: 5 '-gtgctgctgaacatgctgat-3 ',
Downstream primer sequence: 5 '-gcttcgtccttgcaaacttc-3 ';
210>6
<211>
<212>DNA
＜213〉artificial sequence
<220>
＜223〉TRPC6 gene primer sequence is used to prepare people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit
<400>6
Upstream primer sequence: 5 '-cagacaatggcggtcaagtt-3 ',
Downstream primer sequence: 5 '-tggtccacgcattatcttcc-3 ';
210>7
<211>
<212>DNA
＜213〉artificial sequence
<220>
＜223〉TRPC7 gene primer sequence is used to prepare people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit
<400>7
Upstream primer sequence: 5 '-gttaaaaccctgccaaacga-3 ',
Downstream primer sequence: 5 '-tcccagatttccttgcattc-3 ';
210>8
<211>
<212>DNA
＜213〉artificial sequence
<220>
＜223〉IP08 gene primer sequence is used to prepare people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit
<400>8
Upstream primer sequence: 5 '-aaccaaggggtggttcattc-3 ',
Downstream primer sequence: 5 '-ttgccacagctcttcatcct-3 '.
Claims (5)
1. people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit, it comprises box body, in box body, be provided with Sptting plate (1) and positive reference substance (2), it is characterized in that: be provided with some reacting holes (3) in Sptting plate (1), described Sptting plate (1) comprises quantitative fluorescent PCR typical curve Sptting plate (11) and quantitative fluorescent PCR testing sample Sptting plate (12); In quantitative fluorescent PCR typical curve Sptting plate (11), quantitative fluorescent PCR testing sample Sptting plate (12), be provided with goal gene TRPC1～TRPC7 reaction mixture and internal control gene IP08 reaction mixture, described goal gene TRPC1～TRPC7 reaction mixture comprises TRPC1～TRPC7 gene primer sequence, SYBR fluorescence dye, archaeal dna polymerase and reactive ion damping fluid, and described internal control gene IP08 reaction mixture comprises IP08 gene primer sequence, SYBR fluorescence dye, archaeal dna polymerase and reactive ion damping fluid; Described goal gene TRPC1～TRPC7 reaction mixture and internal control gene IP08 reaction mixture are arranged in the reacting hole (3).
2. people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit according to claim 1 is characterized in that:
1) nucleotide sequence of described TRPC1 gene primer sequence is as sequence table SEQ ID №: shown in 1;
2) nucleotide sequence of described TRPC2 gene primer sequence is as sequence table SEQ ID №: shown in 2;
3) nucleotide sequence of described TRPC3 gene primer sequence is as sequence table SEQ ID №: shown in 3;
4) nucleotide sequence of described TRPC4 gene primer sequence is as sequence table SEQ ID №: shown in 4;
5) nucleotide sequence of described TRPC5 gene primer sequence is as sequence table SEQ ID №: shown in 5;
6) nucleotide sequence of described TRPC6 gene primer sequence is as sequence table SEQ ID №: shown in 6;
7) nucleotide sequence of described TRPC7 gene primer sequence is as sequence table SEQ ID №: shown in 7;
8) nucleotide sequence of described I P08 gene primer sequence is as sequence table SEQ ID №: shown in 8.
3. people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit according to claim 1 and 2, it is characterized in that: described quantitative fluorescent PCR typical curve Sptting plate (11), quantitative fluorescent PCR testing sample Sptting plate (12) respectively are provided with 96 reacting holes (3), constitute 96 orifice plates.
4. the preparation method of people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit is characterized in that comprising the steps:
1) prepares box body and be placed in the intravital quantitative fluorescent PCR typical curve of box Sptting plate (11) and quantitative fluorescent PCR testing sample Sptting plate (12), the reacting hole (3) of goal gene TRPC1～TRPC7 and internal control gene IP08 is set by the zone in described quantitative fluorescent PCR typical curve Sptting plate (11) and quantitative fluorescent PCR testing sample Sptting plate (12), and each sample is provided with a repeating hole; Each gene is provided with two no template negative control-NTC;
2) purpose of design gene TRPC1～TRPC7 and internal control gene IP08 primer are measured the efficient and the specificity of described primer;
3) institute is surveyed primer and mix with SYBR fluorescence dye, archaeal dna polymerase and reactive ion damping fluid respectively, constitute goal gene TRPC1～TRPC7 reaction mixture and internal control gene IP08 reaction mixture respectively;
4) goal gene TRPC1～TRPC7 reaction mixture and internal control gene IP08 reaction mixture are added on respectively in aforementioned corresponding reacting hole, constitute goal gene TRPC1～TRPC7 and each Sptting plate of internal control gene IP08 quantitative fluorescent PCR;
5) preparation human brain tissue cDNA is .TRPC1～TRPG7 gene masculine control sample, gets during detection to be added in the reacting hole (3) of typical curve Sptting plate (11) after this sample becomes 5 concentration gradients by 2 times of doubling dilutions, constitutes the gradient reacting hole.
5. the purposes of people's transient receptor potential channel albumen fluorescent quantificationally PCR detecting kit is characterized in that: be used for the analysis and research of the good malignant disease of human TRPC gene-correlation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101988157A CN101948912A (en) | 2010-06-10 | 2010-06-10 | Human transient receptor potential channel protein fluorescence quantitative PCR detection kit and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101988157A CN101948912A (en) | 2010-06-10 | 2010-06-10 | Human transient receptor potential channel protein fluorescence quantitative PCR detection kit and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101948912A true CN101948912A (en) | 2011-01-19 |
Family
ID=43452538
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010101988157A Pending CN101948912A (en) | 2010-06-10 | 2010-06-10 | Human transient receptor potential channel protein fluorescence quantitative PCR detection kit and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101948912A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966309A (en) * | 2013-02-04 | 2014-08-06 | 中国科学院上海生命科学研究院 | Early prediction/diagnosis of AD (Alzheimer disease) by determining TRPC6mRNA (transient receptor potential canonical 6 messenger ribonucleic acid) level in peripheral blood cells |
| CN105108817A (en) * | 2015-09-09 | 2015-12-02 | 通用生物系统（安徽）有限公司 | Manufacturing method for 384 hole solid-phase synthesis reaction device used for nucleic acid synthesis |
| CN108753980A (en) * | 2018-07-26 | 2018-11-06 | 四川大学华西医院 | A kind of kit for screening of the metastatic screening of the small papillary carcinoma of thyroid gland |
| CN116716389A (en) * | 2023-04-20 | 2023-09-08 | 芜湖耄智生物科技有限公司 | Primer group for detecting TRPC6 mRNA level, kit, detection method and application thereof in Alzheimer disease diagnosis |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101555522A (en) * | 2009-05-14 | 2009-10-14 | 浙江大学 | Kit used for detecting candida albicans in intestinal tract by fluorescence quantitative PCR method |
-
2010
- 2010-06-10 CN CN2010101988157A patent/CN101948912A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101555522A (en) * | 2009-05-14 | 2009-10-14 | 浙江大学 | Kit used for detecting candida albicans in intestinal tract by fluorescence quantitative PCR method |
Non-Patent Citations (2)
| Title |
|---|
| 《Journal of Molecular Endocrinology》 20100301 Ming Lu et al. Expression and association of TRPC subtypes with Orai1 and STIM1 in human parathyroid 285-294 1-3 第44卷, * |
| 《Molecular Brain Research》 20021230 Antonio Riccio et al. mRNA distribution analysis of human TRPC family in CNS and peripheral tissue 95-104 1-3 第109卷, 第1-2期 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966309A (en) * | 2013-02-04 | 2014-08-06 | 中国科学院上海生命科学研究院 | Early prediction/diagnosis of AD (Alzheimer disease) by determining TRPC6mRNA (transient receptor potential canonical 6 messenger ribonucleic acid) level in peripheral blood cells |
| WO2014117680A2 (en) * | 2013-02-04 | 2014-08-07 | 中国科学院上海生命科学研究院 | Use of trpc6 mrna levels in peripheral blood cells for early detection/diagnosis of senile dementia |
| WO2014117680A3 (en) * | 2013-02-04 | 2014-09-25 | 中国科学院上海生命科学研究院 | Use of trpc6 mrna levels in peripheral blood cells for early detection/diagnosis of senile dementia |
| US9790551B2 (en) | 2013-02-04 | 2017-10-17 | Aging Wise Biotechnology, Inc. | Use of TRPC6 mRNA levels in peripheral blood cells for early detection/diagnosis of senile dementia |
| CN105108817A (en) * | 2015-09-09 | 2015-12-02 | 通用生物系统（安徽）有限公司 | Manufacturing method for 384 hole solid-phase synthesis reaction device used for nucleic acid synthesis |
| CN105108817B (en) * | 2015-09-09 | 2016-09-14 | 通用生物系统（安徽）有限公司 | A kind of manufacture method of the 384 hole solid phase synthesis devices for nucleic acid synthesis |
| CN108753980A (en) * | 2018-07-26 | 2018-11-06 | 四川大学华西医院 | A kind of kit for screening of the metastatic screening of the small papillary carcinoma of thyroid gland |
| CN108753980B (en) * | 2018-07-26 | 2021-01-01 | 四川大学华西医院 | Screening kit for metastatic screening of thyroid papillary carcinoma |
| CN116716389A (en) * | 2023-04-20 | 2023-09-08 | 芜湖耄智生物科技有限公司 | Primer group for detecting TRPC6 mRNA level, kit, detection method and application thereof in Alzheimer disease diagnosis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Danila et al. | TMPRSS2-ERG status in circulating tumor cells as a predictive biomarker of sensitivity in castration-resistant prostate cancer patients treated with abiraterone acetate | |
| CN107868828A (en) | Detect the specific primer probe composition and kit and detection method in EGFR gene T790M sites | |
| CN105838792A (en) | Primer, probe, kit and method for qualitatively detecting fusion genes of leukemia | |
| WO2017202185A1 (en) | Peripheral blood gene marker for screening benign and malignant small pulmonary nodules and use thereof | |
| CN108315432A (en) | DdPCR technologies detect primer, kit and the detection method of IDH1 R132C genetic mutations | |
| CN101948912A (en) | Human transient receptor potential channel protein fluorescence quantitative PCR detection kit and preparation method and application thereof | |
| CN108441557A (en) | PCA3 and PSA gene detecting kits and its application | |
| CN108949961A (en) | For detecting kit and its screening of adenovirus pneumonia | |
| CN114438214A (en) | Colorectal cancer tumor marker and detection method and device thereof | |
| Alsop et al. | A novel tissue atlas and online tool for the interrogation of small RNA expression in human tissues and biofluids | |
| CN103122387A (en) | Rapid circulating tumor cell (CTCs) fluorescence PCR (Polymerase Chain Reaction) hypersensitivity detection kit and application thereof | |
| CN109790586A (en) | The method of diagnosing and treating Abiraterone acetate-steroid-resistant or responsive type metastatic castration-resistant prostate cancer | |
| JP7187081B2 (en) | Methods for early diagnosis and post-treatment monitoring of breast cancer using liquid biopsy multiplex oncogene biomarkers | |
| CN106319069A (en) | Kit for accurately determining pathogen cells and application of kit | |
| CN110396542A (en) | A kind of LncRNA marker and its application in diabetes | |
| CN106011253B (en) | Primer for detecting PDGFRA gene mutation in microcomponent combines and its application | |
| CN104694534A (en) | Non-small cell lung cancer marker as well as detection method and application thereof | |
| KR102290136B1 (en) | Composition for cell free DNA isolation comprising of alginate-polydopamine-silica complex | |
| Bozhenko et al. | Mammaglobin in peripheral blood and the tumor of breast cancer patients | |
| CN104073432B (en) | Kit for detecting liver cancer marker nucleic acid molecules and detection method of kit | |
| CN111850129A (en) | Primer pair, kit and method for detecting stability of NR21 locus of microsatellite | |
| CN104611446B (en) | A kind of diagnosis of colon cancer test kit based on DNAH5 gene | |
| CN104131103B (en) | AMZ1 gene is preparing the purposes in diagnostic kit | |
| CN108384857A (en) | DdPCR technologies detect primer, kit and the detection method of IDH1 R132H genetic mutations | |
| CN109182514A (en) | Pulmonary cancer diagnosis or transfer diagnosis marker LncRNA Loc729658 and kit and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20110119 |