CN111139296A - Real-time fluorescent quantitative PCR primer and method for detecting transcription level of TGF- β gene of tree shrew - Google Patents
Real-time fluorescent quantitative PCR primer and method for detecting transcription level of TGF- β gene of tree shrew Download PDFInfo
- Publication number
- CN111139296A CN111139296A CN202010163306.4A CN202010163306A CN111139296A CN 111139296 A CN111139296 A CN 111139296A CN 202010163306 A CN202010163306 A CN 202010163306A CN 111139296 A CN111139296 A CN 111139296A
- Authority
- CN
- China
- Prior art keywords
- gene
- tgf
- gapdh
- tree shrew
- tree
- 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
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
-
- 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
-
- 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
Abstract
The invention relates to a primer and a method for detecting real-time fluorescent quantitative PCR of a tree shrew TGF- β gene transcription level, belonging to the technical field of molecular biology, wherein the primer comprises a specific upstream primer and a specific downstream primer of a tree shrew TGF- β gene expression level and a specific upstream primer and a specific downstream primer of a tree shrew GAPDH gene serving as an internal reference gene, the primer can realize the specific amplification of the tree shrew GAPDH gene and the tree shrew TGF- β gene, has no amplification signal to non-target genes in a material, can carry out quantitative detection on the change condition of the tree shrew TGF- β gene transcription level through the primer, has the advantages of simple operation, high repeatability, strong specificity and good sensitivity, and lays the foundation for researching the pathogenesis and treatment scheme of uric acid nephropathy by establishing a tree shrew hyperuricemia chronic model and exploring the influence of blood on the uric acid damage related factor TGF- β gene expression.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a real-time fluorescent quantitative PCR primer and a method for detecting the transcription level of a tree shrew TGF- β gene.
Background
Uric acid is the catabolic end product of purine nucleotides in the body, 80% of which are produced by body cells and 20% are obtained from food. Uric acid is mainly discharged from the body through the kidney and small intestine in the human body. When purine nucleotides are ingested too much or the kidney has a reduced function of excreting uric acid, it will result in a blood uric acid concentration higher than the normal range, called hyperuricemia (hua). Long-term hyperuricemia can cause renal interstitial fibrosis and glomerular sclerosis through oxidative stress, inflammatory reaction and other ways, and further cause pathological changes of the kidney.
The occurrence of a plurality of diseases is controlled by a plurality of related genes, and the detection of the expression of the related genes can possibly further explore the pathogenic mechanism of the diseases, thereby providing a theoretical basis for the development of new drugs and the treatment of the diseases. The experiment selects the gene which is frequently detected in the current nephropathyTGF-βTo make a heuristic for the mechanism of renal injury caused by hyperuricemia.TGF-β(transforming growth factor) as an important biological marker of the clinical progression of chronic kidney disease, is considered to be the most critical cytokine in the mechanisms of glomerulosclerosis and tubulointerstitial fibrosis.TGF-βModulation of various key events in normal development and physiology is widely accepted andTGF- βperturbation of the signal has been implicated in the pathogenesis of connective tissue diseases, fibrosis and cancer.TGF-βUpregulation of expression will result in the expression of various components of the extracellular matrix, accelerating the progressive accumulation of extracellular matrix, ultimately leading to glomerulosclerosis and renal interstitial fibrosis.
Thus detectingTGF-βThe expression level of the gene is of great significance for the study of renal diseases.
Disclosure of Invention
The invention aims to provide a real-time fluorescent quantitative PCR primer and a method for detecting the transcription level of a tree shrew TGF- β geneTGF-βThe expression of the gene is regulated byTGF-βThe research of genes and related diseases lays a foundation.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a real-time fluorescent quantitative PCR method for detecting the transcription level of a TGF- β gene of tree shrew for non-diagnostic purposes comprises the following steps:
step (1), respectively taking total RNA extracted from fresh kidney tissues of healthy and to-be-detected tree shrews as templates, and performing reverse transcription to synthesize a first chain of cDNA of the kidney tissues of the tree shrews;
step (2), establishing tree shrewsGAPDHGenes andTGF-βgene standard curve: diluting the first chain of the kidney tissue cDNA of the healthy tree shrew obtained in the step (1) by Easy dilution gradient, respectively taking the undiluted first chain of the cDNA and the diluted cDNA as templates, carrying out real-time fluorescence quantitative detection, and respectively taking the undiluted first chain of the cDNA and the diluted cDNA as templatesTGF-βF andTGF-βR、GAPDHf andGAPDHr is a specific primer, and real-time fluorescent quantitative PCR amplification is carried out to respectively obtain healthy tree shrewsTGF-βGene, gene,GAPDHA lysis curve and an amplification curve of the gene;
step (3), copy number Log with initial template amount10The logarithm value of (A) is taken as an X axis, and the Ct value is taken as a Y axis to draw, so as to respectively obtain the GAPDH gene,TGF-βA standard curve of the gene;
step (4), the first chains of the cDNA of the to-be-detected tree shrew kidney tissue obtained in the step (1) are respectively replaced byTGF-βF andTGF-βR、GAPDHf andGAPDHr is a specific primer, real-time fluorescent quantitative PCR amplification is carried out, the amplification system and the amplification program are the same as the step (2), and the tree shrews to be detected are respectively obtainedTGF-βGene, gene,GAPDHA lysis curve and an amplification curve of the gene; and (4) calculating according to the standard curve obtained in the step (3) to obtain the tree shrewTGF-βThe level of gene transcription;
saidTGF-βF、TGF-βR、GAPDHF andGAPDHthe sequences of the R primers are as follows:
TGF-βF：5＇-atatcaagcgcagtccccac-3＇；
TGF-βR：5＇-acagttctacgtgctgctcc-3＇；
GAPDHF：5＇-agccccatcaccatcttcc-3＇；
GAPDHR：5＇-aatgagccccagccttctc-3＇。
based on the principle of real-time fluorescence quantitative detection, that is, the Cq value of each template and the logarithm of the initial copy number of the template have a linear relation, the formula is that Cq = -1/lg (1+ Ex) × lgX0+ lgN/lg (1+ Ex) (N is the cycle number of the amplification reaction, X0 is the initial template amount, Ex is the amplification efficiency, and N is the amount of the amplification product when the fluorescence amplification signal reaches the threshold intensity.) the larger the initial copy number, the smaller the Cq value, a standard curve can be made by using a standard with a known initial copy number, wherein the abscissa represents the logarithm of the initial copy number and the ordinate represents the Cq value.
Further, it is preferable that the first strand of the cDNA of the tree shrew kidney tissue in the step (1) is diluted by Easy dilution gradient, and the dilution gradient is 5 times, 25 times, 125 times and 625 times.
Further, it is preferable that the real-time fluorescent quantitative PCR amplification system is as follows:
TABLE 1
A total of 10. mu.L.
Further, preferably, the real-time fluorescent quantitative PCR amplification procedure is: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 10s, and annealing at 60 ℃ for 30s for 40 cycles.
The invention also provides primers for detecting the gene transcription level of the TGF- β gene of the tree shrew, which comprise specific upstream and downstream primers of the expression level of the TGF- β gene of the tree shrew and specific upstream and downstream primers of the GAPDH gene of the tree shrew as an internal reference gene;
wherein, the specific upstream and downstream primer sequences of the tree shrew NF-kB gene expression level are as follows:
TGF-βF：5＇-atatcaagcgcagtccccac-3＇；
TGF-βR：5＇-acagttctacgtgctgctcc-3＇；
the specific upstream and downstream primer sequences of the tree shrew Gapdh gene as the reference gene are as follows:
GAPDHF：5＇-agccccatcaccatcttcc-3＇；
GAPDHR：5＇-aatgagccccagccttctc-3＇。
the invention also provides a kit containing the primer for detecting the transcription level of the TGF- β gene of the tree shrew.
Further, preferably, SYBR Premix Ex Taq is also included
(2x)。
The invention also provides application of the primer and the kit in detecting the transcription level of the TGF- β gene of the tree shrew for non-diagnosis purposes.
When the peak on the dissolution curve is not unique during detection, the pollution exists in the experiment, and the detection needs to be carried out again.
Compared with the prior art, the invention has the beneficial effects that:
the primer of the present invention canRealize to tree shrewGAPDHGene and tree shrewTGF-βThe specific amplification of the gene has no amplification signal to non-target genes in the material, the band is single, and the specificity of the primer is good. The primers of the invention can be used for targeting tree shrewsTGF-βCompared with the traditional semi-quantitative detection, the quantitative detection of the change condition of the gene transcription level can greatly reduce the detection workload, save the labor, the material and the financial resources and quickly obtain the experimental result.
Drawings
FIG. 1 shows tree shrewGAPDHAn amplification curve of the gene;
FIG. 2 shows tree shrewGAPDHA standard curve of the gene;
FIG. 3 shows tree shrewTGF-βAn amplification curve of the gene;
FIG. 4 shows tree shrewTGF-βA standard curve of the gene;
FIG. 5 shows tree shrewGAPDHAn expression amplification curve of the gene;
FIG. 6 shows tree shrewGAPDHExpression melting peak of gene;
FIG. 7 shows tree shrewTGF-βAn expression amplification curve of the gene;
FIG. 8 shows tree shrewTGF-βExpression melting peak of gene;
FIG. 9 shows tree shrewTGF-βRelative expression of the genes;
FIG. 10 shows tree shrewTGF-βGene amplification product gel diagram; m: marker; 1 and 2 are the glue maps of the amplification products of two different tree shrews.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.
The test methods used in the following examples are conventional methods unless otherwise specified.
The materials and reagents used in the following examples were all commercially available unless otherwise specified.
1. Experimental animals used in this example: tree shrews, 9 females and 10 males.
2. Grouping and administration of experimental animals: 1 male tree shrew to-be-taken kidney tissue of 19 tree shrews subjected to blank serum detection is taken out to be subjected to standard curve, and the rest 18 tree shrews are randomly divided into a control group, a 30-day administration group and a 120-day administration group, wherein 6 tree shrews are respectively subjected to the standard curve. The weight of each female is 110-135 g, and the weight of each male is 120-150 g. Intraperitoneal injection (iP) of sodium carboxymethylcellulose solution (CMC-Na, 1g of sodium carboxymethylcellulose dissolved in 100ml of deionized water and 40 mg/kg) with mass concentration of 1% per day in a control group, wherein the administration time is 120 days, the administration group is injected with potassium Oxonate (OA) in the same dose of iP, the administration time is 30 days and 120 days respectively, after 1 hour of fasting administration in the last day, the control group is killed by cervical dislocation, and 0.1g of kidney is rapidly collected and used as a tree shrew kidney injury related factorTGF-βAnd (4) detecting gene expression.
3. Experimental methods
3.1 Collection of Tree shrew Kidney tissue
Kidney tissue was collected and placed in RNA co-solvent (trepe, Roche) for tree shrew kidney injury-associated factorTGF-βAnd (4) detecting gene expression.
3.2 extraction of Total RNA from Kidney
Placing the fresh kidneys of 19 tree shrews in a high-flux tissue pulverizer to pulverize for 300 seconds at 60Hz, taking out and standing for 5 min; and then transferring the mixture into a 1.5mL EP tube for standing for 5min, adding 200 mu L of chloroform precooled at the temperature of-20 ℃, fully oscillating in a vortex, standing for 15min, centrifuging at the temperature of 4 ℃ and 12000r/min for 25min, sucking 450 mu L of supernate into another 1.5mL EP tube, adding isopropanol precooled at the temperature of-20 ℃ in an equal volume, fully mixing and standing for 10min, centrifuging at the temperature of 4 ℃ and 12000r/m for 10min, removing the supernate, slightly drying the inner wall of the tube, adding 75% ethanol with precooled volume percentage concentration of 1 mL-20 ℃ for washing precipitate, centrifuging at the temperature of 4 ℃ and 7500r/m for 5min, removing the supernate, slightly drying the inner wall of the tube, adding 30 mu L of DEPC for dissolving precipitate, and measuring the RNA concentration of the extracted RNA by using an ultramicro spectrophotometer, wherein the values of A260nm/A280nm and A260nm/A230nm are obtained. Diluting the extracted RNA to 1000 ng/. mu.L with DEPC water, Calmension x V left = Callution x (V left + V added) (where Calmension: original concentration of extracted RNA; Vleft: original volume of extracted RNA; Callution: 1000 ng/. mu.L; Vadd: volume of DEPC water required for dilution)
3.3 Synthesis of cDNA
The extracted kidney RNA was manipulated according to the instructions of the reverse transcription Kit Prime script RT Reagent Kit, and the system was 30. mu.L. The reaction system composition is shown in Table 2.
TABLE 2
The reverse transcription conditions were: the cDNA is obtained by reverse transcription at 37 ℃ for 15min, 85 ℃ for 5s and 4 ℃ for 10 min. The reverse transcription product cDNA was stored at-20 ℃ for future use.
3.4 design of primers for Gene of interest
According to tree shrews in NCBI gene bankTGF-βAndGAPDHthe gene sequences are respectively subjected to primer design by using primer design software Pimerexpress 5.0, synthesized by Beijing Baitach company,GAPDHused as an internal reference gene.
3.5 the primer sequences and fragment sizes for quantifying the expression level of the TGF- β gene of the tree shrew of this example are shown in Table 3
TABLE 3
3.6 determination of fluorescent quantitative PCR reaction System of target Gene
Amplification and data analysis were performed on a Real-Time fluorescence quantitative analyzer CFX96 Real-Time System according to TAKARA Bio Inc PCR reagent (SYBR Premix Ex TaqII) in the PCR amplification System shown in Table 4:
TABLE 4
A total of 10. mu.L.
The real-time fluorescent quantitative PCR amplification program comprises the following steps: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 10s, and annealing at 60 ℃ for 30s for 40 cycles.
Wherein the nucleotide sequence of the amplified fragment of the tree shrew TGF- β gene is shown as SEQ ID NO.5, and the nucleotide sequence of the amplified fragment of the tree shrew internal reference gene GAPDH is shown as SEQ ID NO. 6.
3.7 Tree shrewGAPDHEstablishing a gene and TGF- β gene standard curve, namely, diluting a first chain of rat kidney cDNA which is synthesized by reverse transcription in an easy dilution gradient step 3.3 and is not administrated by 5 times, 25 times, 125 times and 625 times, respectively taking the first chain of original cDNA and the diluted cDNA as templates, respectively carrying out real-time fluorescence quantitative detection on 2 parallel samples, carrying out real-time fluorescence quantitative PCR according to a step 3.6 fluorescence quantitative PCR system, and carrying out the principle of real-time fluorescence quantitative detection, namely, the Cq value of each template and the logarithm of the initial copy number of the template have a linear relation, wherein the formula is that the more Ct = -1/lg (1+ Ex) = lgX0+ lgN/lg (1+ Ex) (N is the cycle number of amplification reaction, X0 is the initial template amount, Ex is the amplification efficiency, and the more the initial copy number of an amplification product when N is the fluorescence amplification signal reaches the threshold intensity), the smaller the Cq value is based on the principle of the BioRad 3.3 and the TGF-539 gene standard curve can be obtained by using the BioRad 3.3.3.3.3 gene and the TGF-PDH standard curve.
3.8 analysis of Gene expression differences in samples
According to the TGF- β gene and GAPDH gene standard curve obtained in step 3.7 and the Cq value of each sample, the tree shrew TGF- β gene transcription level can be obtained by utilizing the self-carried gene expression calculation function in the Bio-Rad CFX Manager 3.1 software.
4 results
4.1 Tree shrewTGF-βMelting curve of gene fluorescent quantitative PCR reaction
As can be seen from fig. 8 and 10, inTGF-βThe dissolution curve of the fluorescence quantitative PCR reaction in the gene amplification process only shows a single peak, and the reaction productThe electrophoresis result of the substance only sees a specific expression band, which shows that the specificity of the amplified target fragment is good.
4.2 Tree shrewTGF-βGenes andGAPDHstandard curve of gene
As can be seen from FIGS. 2 and 4, the tree shrewTGF-βGenes andGAPDHstandard Curve R of Gene2All are close to 1, which indicates that the relative quantification performed by the standard curve is more accurate, and because the fluorescence intensity is stronger, the relative synchronization between the increase of the fluorescence intensity and the amplification of the PCR can be ensured, and the expression of the PCR can be accurately detected, so as toGAPDHChanges in mRNA expression levels were obtained as endogenous controls.
4.3 hyperuricemia Tree shrew kidney tissue caused by Potassium OxonateTGF-βmRNAQuantitative detection of changes in expression levels
2, reverse transcription of RNA extracted from the fresh kidney tissues obtained from each group to obtain cDNA, and real-time fluorescence quantitative PCR detection to obtain each groupTGF-βmRNA expression level difference, injecting potassium oxonate with 40mg/kg dose into abdominal cavity,TGF-βin the kidney mRNA expression level, TGF- β in the kidney mRNA expression level was 1.3 in the control group, 1.0 in the 30-day group, and was down-regulated compared to the control group, 1.9 in the 120-day group, and was up-regulated 1.5-fold compared to the control group, as shown in FIG. 9.
4.4 Potassium Oxonate (OA) is a uricase inhibitor, which can reduce the decomposition and excretion of uric acid by inhibiting uricase activity to increase uric acid level, and can cause hyperuricemia in Tree shrew, and is frequently used in primate modeling, and can be used in the above examplesTGF-βmRNA expression is first down-regulated and then up-regulated over time of administration; illustrating that the invention can be usedTGF-βDetection of mRNA expression level for the study of Tree shrewTGF-βThe function and the influencing factors of the gene provide effective tools and provide reliable means for the research of diseases such as hyperuricemia and the like.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
SEQ ID NO.1
agccccatca ccatcttcc19
SEQ ID NO.2
aatgagcccc agccttctc 19
SEQ ID NO.3
SEQ ID NO.4
SEQ ID NO.5
atatcaagcg cagtccccac agcatataca tgctctttaa cacgtcagag ctccgagaag 60
cggtgcccga ccctctgttg ctctcccggg cagagctgcg cctactgagg ctcaagttaa 120
aggtggagca gcacgtagaa ctgt 144
SEQ ID NO.6
ggcacagtca aggctgagaa tgggaagctg gtcatcaacg ggaaacccat caccatcttc 60
caggagcgag atcccgctaa catcaaatgg ggtgatgctg gtgctgagta tgtcgtggag 120
tctactggcg tcttcaccac cat 143
Sequence listing
<110> institute of medical science and biology of China academy of medical sciences
<120> primers and method for real-time fluorescent quantitative PCR for detecting transcription level of TGF- β gene of tree shrew
<160>6
<170>SIPOSequenceListing 1.0
<210>1
<211>19
<212>DNA
<213> Artificial sequence ()
<400>1
agccccatca ccatcttcc 19
<210>2
<211>19
<212>DNA
<213> Artificial sequence ()
<400>2
aatgagcccc agccttctc 19
<210>3
<211>20
<212>DNA
<213> Artificial sequence ()
<400>3
<210>4
<211>20
<212>DNA
<213> Artificial sequence ()
<400>4
<210>5
<211>144
<212>DNA
<213> Artificial sequence ()
<400>5
atatcaagcg cagtccccac agcatataca tgctctttaa cacgtcagag ctccgagaag 60
cggtgcccga ccctctgttg ctctcccggg cagagctgcg cctactgagg ctcaagttaa 120
aggtggagca gcacgtagaa ctgt 144
<210>6
<211>143
<212>DNA
<213> Artificial sequence ()
<400>6
ggcacagtca aggctgagaa tgggaagctg gtcatcaacg ggaaacccat caccatcttc 60
caggagcgag atcccgctaa catcaaatgg ggtgatgctg gtgctgagta tgtcgtggag 120
tctactggcg tcttcaccac cat 143
Claims (8)
1. A real-time fluorescent quantitative PCR method for detecting the transcription level of a TGF- β gene of tree shrew for non-diagnostic purposes is characterized by comprising the following steps:
step (1), respectively taking total RNA extracted from fresh kidney tissues of healthy and to-be-detected tree shrews as templates, and performing reverse transcription to synthesize a first chain of cDNA of the kidney tissues of the tree shrews;
step (2), establishing tree shrewsGAPDHGenes andTGF-βgene standard curve: diluting the first chain of the kidney tissue cDNA of the healthy tree shrew obtained in the step (1) by Easy dilution gradient, respectively taking the undiluted first chain of the cDNA and the diluted cDNA as templates, carrying out real-time fluorescence quantitative detection, and respectively taking the undiluted first chain of the cDNA and the diluted cDNA as templatesTGF-βF andTGF-βR、GAPDHf andGAPDHr is a specific primer, and real-time fluorescent quantitative PCR amplification is carried out to respectively obtain healthy tree shrewsTGF-βGene, gene,GAPDHA lysis curve and an amplification curve of the gene;
step (3), copy number Log with initial template amount10The logarithm value of (A) is taken as an X axis, and the Ct value is taken as a Y axis to draw, so as to respectively obtain the GAPDH gene,TGF-βA standard curve of the gene;
step (4), the first chains of the cDNA of the to-be-detected tree shrew kidney tissue obtained in the step (1) are respectively replaced byTGF-βF andTGF-βR、GAPDHf andGAPDHr is a specific primer, real-time fluorescent quantitative PCR amplification is carried out, the amplification system and the amplification program are the same as the step (2), and the tree shrews to be detected are respectively obtainedTGF-βGene, gene,GAPDHA lysis curve and an amplification curve of the gene; and (4) calculating according to the standard curve obtained in the step (3) to obtain the tree shrewTGF-βThe level of gene transcription;
saidTGF-βF、TGF-βR、GAPDHF andGAPDHthe sequences of the R primers are as follows:
TGF-βF：5＇-atatcaagcgcagtccccac-3＇；
TGF-βR：5＇-acagttctacgtgctgctcc-3＇；
GAPDHF：5＇-agccccatcaccatcttcc-3＇；
GAPDHR：5＇-aatgagccccagccttctc-3＇。
2. the method for real-time fluorescent quantitative PCR for detecting the transcription level of the TGF- β gene of tree shrew for non-diagnostic purposes according to claim 1, wherein the first strand of the kidney tissue cDNA of tree shrew in step (1) is diluted by Easy dilution gradient, wherein the dilution gradient is 5 times, 25 times, 125 times and 625 times respectively.
3. The method for detecting real-time fluorescent quantitative PCR of the transcription level of the TGF- β gene of tree shrew for non-diagnostic purposes according to claim 1, wherein the real-time fluorescent quantitative PCR amplification system is as follows:
a total of 10. mu.L.
4. The method for detecting the real-time fluorescent quantitative PCR of the transcription levels of the TGF- β genes of tree shrews for the non-diagnostic purpose according to claim 1, wherein the real-time fluorescent quantitative PCR amplification program comprises 40 cycles of pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 10s and annealing at 60 ℃ for 30 s.
5. The primers for detecting the gene transcription level of the TGF- β gene of the tree shrew are characterized by comprising specific upstream and downstream primers of the expression level of the TGF- β gene of the tree shrew and specific upstream and downstream primers of the GAPDH gene of the tree shrew as an internal reference gene;
wherein, the specific upstream and downstream primer sequences of the tree shrew NF-kB gene expression level are as follows:
TGF-βF：5＇-atatcaagcgcagtccccac-3＇；
TGF-βR：5＇-acagttctacgtgctgctcc-3＇；
the specific upstream and downstream primer sequences of the tree shrew Gapdh gene as the reference gene are as follows:
GAPDHF：5＇-agccccatcaccatcttcc-3＇；
GAPDHR：5＇-aatgagccccagccttctc-3＇。
6. a kit containing the primers for detecting the transcription level of the TGF- β gene of tree shrew according to claim 1.
7. The kit of claim 6, further comprising SYBR Premix Ex Taq
(2x)。
8. Use of the primers of claim 5 and the kit of claim 6 or 7 for detecting the transcription level of the TGF- β gene of tree shrew for non-diagnostic purposes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010163306.4A CN111139296A (en) | 2020-03-10 | 2020-03-10 | Real-time fluorescent quantitative PCR primer and method for detecting transcription level of TGF- β gene of tree shrew |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010163306.4A CN111139296A (en) | 2020-03-10 | 2020-03-10 | Real-time fluorescent quantitative PCR primer and method for detecting transcription level of TGF- β gene of tree shrew |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111139296A true CN111139296A (en) | 2020-05-12 |
Family
ID=70528491
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010163306.4A Pending CN111139296A (en) | 2020-03-10 | 2020-03-10 | Real-time fluorescent quantitative PCR primer and method for detecting transcription level of TGF- β gene of tree shrew |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111139296A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039248A (en) * | 2015-05-24 | 2015-11-11 | 昆明学院 | Tree shrew mesenchymal stem cells culture system |
| CN108330172A (en) * | 2017-09-29 | 2018-07-27 | 中国医学科学院医学生物学研究所 | The method that RT-qPCR detects tree shrew SLC22A12/URAT1 gene transcription levels |
| CN111226865A (en) * | 2020-03-02 | 2020-06-05 | 云南省第二人民医院 | Method for constructing tree shrew choroidal neovascularization animal model |
-
2020
- 2020-03-10 CN CN202010163306.4A patent/CN111139296A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039248A (en) * | 2015-05-24 | 2015-11-11 | 昆明学院 | Tree shrew mesenchymal stem cells culture system |
| CN108330172A (en) * | 2017-09-29 | 2018-07-27 | 中国医学科学院医学生物学研究所 | The method that RT-qPCR detects tree shrew SLC22A12/URAT1 gene transcription levels |
| CN111226865A (en) * | 2020-03-02 | 2020-06-05 | 云南省第二人民医院 | Method for constructing tree shrew choroidal neovascularization animal model |
Non-Patent Citations (2)
| Title |
|---|
| ANDREW IAN JOBLING等: "Retinal and choroidal TGF-β in the tree shrew model of myopia: isoform expression, activation and effects on function", 《EXP EYE RES》 * |
| 杨淑芬等: "高尿酸血症的中药治疗机制研究进展", 《中国中医急症》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Small et al. | Stability of reference genes for real-time PCR analyses in channel catfish (Ictalurus punctatus) tissues under varying physiological conditions | |
| Johansen et al. | Differences in gene expression in Atlantic salmon parr and smolt after challenge with Piscine orthoreovirus (PRV) | |
| CN101619346B (en) | Method for detecting copy number variation of human coronary heart disease susceptibility gene-lipoprotein a gene and kit thereof | |
| Chen et al. | An evaluation of hepatic glucose metabolism at the transcription level for the omnivorous GIFT tilapia, Oreochromis niloticus during postprandial nutritional status transition from anabolism to catabolism | |
| Yan et al. | microRNA expression signature in skeletal muscle of Nile tilapia | |
| CN108330172A (en) | The method that RT-qPCR detects tree shrew SLC22A12/URAT1 gene transcription levels | |
| CN106119393B (en) | Plasma miRNA marker related to esophageal squamous carcinoma auxiliary diagnosis and application thereof | |
| US20190153446A1 (en) | Mir-149-3p and method for treating metabolic disease using the same | |
| Xu et al. | Characterization of grass carp spleen transcriptome during GCRV infection | |
| CN109082470A (en) | The two generation sequencing primer probe groups and its detection method of microsatellite instability state | |
| CN111088361A (en) | Long-chain non-coding RNA marker for early diagnosis of malignant melanoma and application thereof | |
| CN111778278B (en) | Construction method and application of Slfn 4-deleted atherosclerosis model mouse | |
| CN111139296A (en) | Real-time fluorescent quantitative PCR primer and method for detecting transcription level of TGF- β gene of tree shrew | |
| CN105969897A (en) | Chronic kidney disease renal function diagnostic kit based on lncRNA-H19 and application of lncRNA-H19 | |
| Lu et al. | The function of miR-122 in the lipid metabolism and immunity of grass carp (Ctenopharyngodon idellus) | |
| CN109593852B (en) | Serum miRNA marker related to nasopharyngeal carcinoma auxiliary diagnosis and application thereof | |
| CN109913458B (en) | circRNA and application thereof in detecting hypoxic-ischemic brain injury | |
| CN107459546A (en) | A kind of tRF Gly antisenses chain inhibitor and its application | |
| CN111187846A (en) | Primer, kit and method for detecting tree shrew LCN2 gene transcription level | |
| CN109402228A (en) | The method of RT-qPCR detection tree shrew SLC2A9/Glut9 gene transcription level | |
| CN111197084A (en) | Primer, kit and method for detecting transcription level of NF-kB gene of tree shrew | |
| CN111154868A (en) | Method for detecting tree shrew SLC22A6 gene transcription level by real-time fluorescence quantitative PCR method, primer and kit | |
| CN111172251A (en) | Method, kit and primer for detecting tree shrew SLC22A8 gene transcription level by RT-qPCR method | |
| Yang et al. | Evaluation of qPCR reference genes for taimen (Hucho taimen) under heat stress | |
| CN106119396B (en) | Plasma miRNA marker related to hashimoto thyroiditis auxiliary diagnosis and application thereof |
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 |