CN106498042A - The method that RT qPCR detect Wistar rat xanthine dehydrogenases/lysyloxidase gene transcriptional level - Google Patents
The method that RT qPCR detect Wistar rat xanthine dehydrogenases/lysyloxidase gene transcriptional level Download PDFInfo
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Abstract
The present invention discloses a kind of method that RT qPCR detect Wistar rat xanthine dehydrogenases/lysyloxidase gene transcriptional level, designs primer, and as template, routinely reverse transcription synthesizes to obtain the first chains of rat liver tissue cDNA to the total serum IgE with rat fresh liver tissue extraction；Rat GAPDH genes and XDH/XO gene standard curves is set up, the total serum IgE that rat liver is extracted is collected fluorescence through real-time fluorescence quantitative PCR detection and compared with gained dissolving peak standard curve, that is, obtain Wistar rats xanthine dehydrogenase/lysyloxidase gene transcriptional level.Detection by quantitative can be implemented to the situation of change of rat xanthine dehydrogenase/lysyloxidase gene transcriptional level by the primer of the present invention, which is simple to operate, repeatable high, high specificity, sensitivity are good.The present invention provides effective tool for the function and influence factor for studying rat xanthine dehydrogenase/lysyloxidase gene.
Description
Technical field
The present invention relates to a kind of detection method, is detected with real time fluorescent quantitative RT-qPCR methods more particularly, to a kind of
The method of Wistar rats xanthine dehydrogenase/lysyloxidase gene transcriptional level, belongs to field of molecular biotechnology.
Background technology
Xanthine dehydrogenase/oxidase (XDH/XO) is a kind of important enzyme in nucleic acid in vivo metabolism, in purine metabolism mistake
The last two steps of purine metabolism are catalyzed in journey, form uric acid metabolism end-product uric acid from xanthine and hypoxanthine.The enzyme is extensive
It is distributed in various animals and mankind's body, is primarily present in liver.
Hyperuricemia is caused by urate deposition or uric acid metabolism obstacle serum uric acid level increase
A kind of disease, in recent years sickness rate constantly rise.Research show its not still gout outbreak pathologic basis, also with diabetes,
The diseases such as hypertension, metabolism disorder syndrome, insuline resistance syndrome have close relationship, and animal model is used as study of disease
Pathogenesis and a kind of pharmacologic agent method have very important effect in scientific development.Inquire into sending out for hyperuricemia
Anttdisease Mechanism and to set up animal model particularly important for hyperuricemia is captured.
Its structure, sequence, work(are concentrated mainly on to the research of xanthine dehydrogenase/oxidase (XDH/XO) in document report
On energy and mortifier, increasingly pay attention to recently the research of its transcriptional level, mice, the detection method of tree have all been studied.
The gene amplification method of real-time fluorescence quantitative PCR has been widely used in the quantitative study of gene transcription level.The party
Method has the advantages that high, reproducible high specificity, sensitivity, quantitative accurate, high degree of automation, totally-enclosed reaction.
For studying the transcriptional level of xanthine dehydrogenase/oxidase (XDH/XO) in rat body, need to set up one kind being capable of essence
Really the method to xanthine dehydrogenase/oxidase (XDH/XO) mRNA detection by quantitative, is rat xanthine dehydrogenase/oxidase
(XDH/XO) further research lays the first stone.
Content of the invention
It is an object of the invention to provide one kind real time fluorescent quantitative RT-qPCR methods detection rat xanthine dehydrogenase/
The method of lysyloxidase gene transcriptional level, to carry out detection by quantitative to xanthine dehydrogenase/lysyloxidase gene on transcriptional level.
For achieving the above object, the present invention is completed by following technical proposal：With the reverse transcription of sample total serum IgE into cDNA be
Template, carries out real-time fluorescence quantitative PCR amplification using PCR primer combination, quantitative according to the situation of change of fluorescence in reaction system
Xanthine dehydrogenase/lysyloxidase gene transcriptional level.
The present invention concrete scheme be：A kind of RT-qPCR detections Wistar rat xanthine dehydrogenases/lysyloxidase gene turns
The method of record level, it is characterised in that comprise the following steps：
(1) following primer is designed：
The specificity upstream and downstream primer combination of rat XDH/XO gene expression doses and corresponding nucleotides sequence are classified as：
XDH/XO F：5 ＇-ATGCGGACCCTGAAACAACA-3 ＇
XDH/XO R：5 ＇-TGTTCTGAAGACGGTCATACTTGGA-3 ＇；
The primer combination of specificity upstream and downstream and corresponding nucleotide sequence as the rat GAPDH genes of reference gene
For：
GAPDH F：5 ＇-GGCACAGTCAAGGCTGAGAATG-3 ＇
GAPDH R：5 ＇-ATGGTGGTGAAGACGCCAGTA-3 ＇；
(2) as template, routinely reverse transcription synthesizes to obtain rat liver tissue to the total serum IgE with rat fresh liver tissue extraction
The first chains of cDNA；
(3) rat GAPDH genes and XDH/XO gene standard curves are set up：With Esidilution gradient dilution steps (2)
The first chains of rat liver tissue cDNA, are diluted to 10 respectively-1、10-2、10-3、10-4Times concentration, with the first chain of original cDNA and dilute
CDNA after releasing is template, carries out real time fluorescent quantitative detection, with the XDH/XO F and XDH/XO R in step (1) and
GAPDH F and GAPDH R is specific primer, respectively under following PCR amplification system：25 μ L systems, SYBR Premix Ex
Taq II (Tli RNaseH Plus) 12.5 μ L, positive, each 1 μ L of reverse primer, 1 μ L of cDNA templates, 9.5 μ L of deionized water, enter
The following real-time fluorescence quantitative PCR amplification of row：94 DEG C of denaturations 30s, 94 DEG C of degeneration 5s, 59 DEG C of annealing 30s, 40 circulations, 59 DEG C
First order fluorescence is gathered to 94 DEG C per 5s, obtain the solubility curve and standard curve of rat GAPDH genes and XDH/XO genes；
(4) total serum IgE that rat liver is extracted is collected fluorescence through real-time fluorescence quantitative PCR detection and is dissolved with step (3) gained
Peak standard curve compares, that is, obtain Wistar rats xanthine dehydrogenase/lysyloxidase gene transcriptional level.
Advantage and effect that the present invention possesses：The present invention is detected suitable for real-time fluorescence quantitative PCR.Application present invention institute is public
The primer sequence that opens is can achieve to rat GAPDH genes and the specific amplification of XDH/XO genes, to non-genes of interest in material
Without amplified signal, can be to the change feelings of rat xanthine dehydrogenase/lysyloxidase gene transcriptional level by the primer of the present invention
Condition implements detection by quantitative, and which is simple to operate, and repeatable high, high specificity, sensitivity are good.The present invention is research rat xanthine
The function of dehydrogenase/lysyloxidase gene and influence factor provide effective tool.
Description of the drawings
Agarose gel electrophoresis figures of the Fig. 1 for rat RNA integrity mensuration's；
Fig. 2 is rat XDH/XO gene solubility curves；
Fig. 3 is rat XDH/XO mRNA RT-qPCRc product agarose gel electrophoresis figures；
Standard curves of the Fig. 4 for rat GAPDH genes；
Standard curves of the Fig. 5 for rat XDH/XO genes；
Fig. 6 changes for rat mRNA XDH gene expression doses.
Specific implementation method
Test method used in following embodiments, if no special instructions, is conventional method.
Material agents used in following embodiments etc., if no special instructions, are commercial sources and obtain.
1st, the laboratory animal used in the present embodiment：Cleaning grade Wistar rats, female, 40.
2nd, the packet and administration of laboratory animal：The female rats 40 of health, are randomly divided into 5 groups, 8/group, first group of abdomen
The Oteracil Potassium (OA) of chamber injection 300mg/kg dosage 37.5mg/ml concentration is used as OA groups, second group of lumbar injection 22mg/kg agent
Amount 2.75mg/ml allopurinol (ALLO) as ALLO groups, the CMC-Na of the 3rd group of lumbar injection 1% as a control group, the 4th
The allopurinol of group lumbar injection 300mg/kg dosage Oteracil Potassium and 22mg/kg dosage as OA+ALLO22mg/kg groups, the 5th
The allopurinol of group lumbar injection 300mg/kg dosage Oteracil Potassium and 3mg/kg dosage 0.4mg/ml concentration is used as OA+ALLO3mg/
Kg, between every rat, administration time is spaced 10 minutes, and in 2 rats of every group of random choose, puts to death and respectively take after 1.5h is administered
Liver 0.1g, for the detection of xanthine dehydrogenase/oxidase (XDH/XO) gene transcription level.
3rd, experimental technique
The collection of 3.1 rat liver tissues
Separate and win liver organization RNA cosolvents (Tripure, Roche company) are placed in for xanthine dehydrogenase/oxygen
Change the detection of enzyme (XDH/XO) gene transcription level.
The extraction of 3.2 liver total RNA
Fresh liver 0.1g is taken in homogenizer, adds 1ml tripure to be fully homogenized at room temperature, stand 5min, then
Standing 5min in 1.5mlEP pipes is transferred them to, adds the chloroform of 200ul-20 DEG C of pre-cooling, whirlpool concussion fully, to stand 15min
4 DEG C afterwards, 12000r/m centrifugation 25min, in another 1.5mlEP pipes, equal-volume adds -20 DEG C in advance to Aspirate supernatant 450ul
Cold isopropanol, fully mixes and stands 10min, 4 DEG C, 12000r/m centrifugation 10min, abandons supernatant, treats that inside pipe wall is slightly dry, plus
The 75% washing with alcohol precipitation of 1ml-20 DEG C of pre-cooling, 4 DEG C, 7500r/m centrifugation 5min, abandons supernatant, treats that inside pipe wall is slightly dry, add
30ulDEPC water dissolutioies are precipitated, and 65 DEG C of water-bath 10min, the total serum IgE sample for being extracted take 1ul through Nanodrop-1000 ultramicron
Nucleic acid determination instrument determines concentration and absorbance ratio.Add DEPC water to be diluted to 1000ng/ul after determining concentration, be put into -80 DEG C of ice
Case is standby.
3.3RNA integrity detection
Taking the 2 μ l of total serum IgE sample extracted by 3.2 steps at random carries out 1.5% agarose gel electrophoresiies (120V, 400mA)
20min, observes 28S under gel imaging system, and the integrity of RNA is analyzed in 18S and 5S band brightness.As a result can be observed
Tri- band of 28S, 18S and 5S, is shown in Fig. 1, and extracted RNA is described without degraded, and integrity is good, can be used for subsequent experimental.
The synthesis of 3.4cDNA
According to Reverse Transcriptase kit PrimeScriptTMRT reagent Kit explanation operations, add in every 10 μ l systems successively
Enter 5 × PrimeScript Buffer, 2 μ l, 0.5 μ l of PrimeScript RT Enzyme Mix, Oligo dT Primer
0.5 μ l of 0.5 μ l, Random 6mers, total serum IgE (concentration dilution is 1000ng/ μ l) 1 μ l, 5.5 μ l of R NaseFree dH2O,
Reverse transcription condition is:37 DEG C, 15min, 85 DEG C, 5s, 4 DEG C, 10min.
The design of 3.5 genes of interest primers
According to the gene order of the XDH/XO and GAPDH of rat in NCBI gene banks, using primer-design software Pimer
Express 5.0 carries out design of primers respectively, synthesizes through hundred Imtech of Beijing, and GAPDH is used as reference gene.
3.6 the present embodiment rat XDH/XO gene expression doses are quantitatively shown in Table 1 with primer sequence and clip size.
Table 1
The determination of 3.7 genes of interest quantitative fluorescent PCR reaction systems
TAKARA biologies company limited's product PCR reagent (SYBR Premix Ex TaqII) is pressed, in real time fluorescent quantitative
Expanded and data analysiss on instrument CFX96Real-Time System, PCR amplification system is as follows：XDH and GAPDH upstream and downstream
The each 1ul of primer (10p), SYBR Premix Ex Taq II (Tli RNaseH Plus) 12.5ul, sterile deionized water
9.5ul、cDNA 1ul；Carry out following PCR amplifications：94 DEG C of denaturations 30s, 94 DEG C of degeneration 5s, 59 DEG C of annealing 30s, 40 circulations,
59 DEG C to 94 DEG C gather first order fluorescence per 5s.
3.8 rat GAPDH genes and the foundation of XDH/XO gene standard curves：With Esidilution gradient dilution steps
First chains of rat liver cDNA of 3.4 reverse transcriptions synthesis, are diluted to 10 respectively-1、10-2、10-3、10-4Times concentration, with original cDNA
CDNA after first chain and dilution is template, and respectively doing 2 Duplicate Samples carries out real time fluorescent quantitative detection, fixed by step 3.7 fluorescence
Amount PCR system carries out real-time fluorescence quantitative PCR, obtains the solubility curve and standard curve of XDH/XO genes and GAPDH genes.
3.9 sample gene differential expressions are analyzed
The copy number that each dilution gradient cDNA can be obtained by standard curve ct values, correspondingly obtains genes of interest with sample ct values
Relative expression quantity.
4 results
The solubility curve of 4.1 rat XDH/XO gene by fluorescence quantitative PCR reactions
From Fig. 2 and Fig. 3, the rarely seen list of the solubility curve of quantitative fluorescent PCR reaction in XOD/XO gene amplification process
Only peak, the rarely seen specially band of the electrophoresis result of product, shows that the purpose fragment specificity for expanding is good.
4.2 rat XDH/XO genes and the standard curve of GAPDH genes
Standard curve R from Fig. 4 and Fig. 5, rat XOD/XO genes and GAPDH genes21 is close to, is illustrated with this
It is more accurate that standard curve carries out relative quantification, as fluorescence intensity is relatively strong, therefore can guarantee that the increase of fluorescence intensity and the expansion of PCR
Increase relative synchronization, can accurately detect the expression of PCR, with GAPDH as endogenous control thing, obtain the change of mRNA expressions.
4.3 allopurinols to Oteracil Potassium caused by hyperuricemia rat liver tissue XDH/XO mRNA expressions
The detection by quantitative of change
The fresh liver obtained by step 2 each group organizes extracted RNA, and reverse transcription obtains cDNA, through real time fluorescent quantitative
PCR detections, can obtain each group XDH/XO mRNA expression differences, the Oteracil Potassium of lumbar injection 300mg/kg dosage, liver group
MRNA XDH gene expressions rise is knitted, injection allopurinol gene expression is lowered relative to matched group, and injection Oteracil Potassium is noted simultaneously
The rise of allopurinol group gene expression relative comparison group is penetrated, is lowered with respect to Oteracil Potassium OA group gene expressions.See Fig. 6.
4.4 Oteracil Potassiums (OA) are uricase inhibitors, can pass through to suppress uricase activity, reduce decomposition and the row of uric acid
Let out, raise uric acid level, mice, rat hyperuricemia can be caused, with more in rodent modeling, for above-mentioned
Embodiment can make XDH/XO mRNA up-regulateds；Allopurinol (ALLO) gives birth to uric acid by the activity for suppressing xanthine oxidase
Into minimizing, the uric acid content in blood and in urine is reduced to dissolubility level below, is mainly used in treating gout and prevents gout
The auxiliary treatment of property nephropathy, secondary hyperuricemia and serious symptom epilepsy, can make XDH/XO mRNA tables for above-described embodiment
Up to downward.Impact result of the experimental result of step 4.3 with Oteracil Potassium and allopurinol to rat is consistent, illustrates that the present invention is available
In the detection of XDH/XO mRNA expressions, the function and influence factor for studying rat XDH/XO genes provides effective work
Tool, the research for diseases such as hyperuricemias provide reliable means.
XDH/XO gene specific forward primer：
XDH/XO F：5 ＇-ATGCGGACCCTGAAACAACA-3 ＇
XDH/XO gene specific downstream primers：
XDH/XO R：5 ＇-TGTTCTGAAGACGGTCATACTTGGA-3 ＇
Reference gene GAPDH specific forward primers：
GAPDH F：5 ＇-GGCACAGTCAAGGCTGAGAATG-3 ＇
Reference gene GAPDH specific Down Stream primers：
GAPDH R：5 ＇-ATGGTGGTGAAGACGCCAGTA-3 ＇
The nucleotide sequence of the rat XDH/XO genetic fragments for being expanded：
atgcggacc ctgaaacaac acttctggtc tacctgagaa gaaagttggg gctatgtggg
accaagcttg gctgtggaga aggtggctgt ggggcatgca ccgtgatgat ctccaagtat gaccgtcttc
agaaca
The nucleotide sequence of the rat reference gene GAPDH fragments for being expanded：
ggcacagtc aaggctgaga atgggaagct ggtcatcaac gggaaaccca tcaccatctt
ccaggagcga gatcccgcta acatcaaatg gggtgatgct ggtgctgagt atgtcgtgga gtctactggc
gtcttcacca ccat
Claims (1)
1. a kind of method that RT-qPCR detects Wistar rat xanthine dehydrogenases/lysyloxidase gene transcriptional level, its feature exist
In comprising the following steps：
（1）Design following primer：
The specificity upstream and downstream primer combination of rat XDH/XO gene expression doses and corresponding nucleotides sequence are classified as：
XDH/XO F：5 ＇-ATGCGGACCCTGAAACAACA-3 ＇
XDH/XO R：5 ＇-TGTTCTGAAGACGGTCATACTTGGA-3 ＇；
The primer combination of specificity upstream and downstream and corresponding nucleotides sequence as the rat GAPDH genes of reference gene is classified as：
GAPDH F：5 ＇-GGCACAGTCAAGGCTGAGAATG-3 ＇
GAPDH R：5 ＇-ATGGTGGTGAAGACGCCAGTA-3 ＇；
（2）As template, routinely reverse transcription synthesizes to obtain rat liver tissue cDNA to total serum IgE with rat fresh liver tissue extraction
First chain；
（3）Set up rat GAPDH genes and XDH/XO gene standard curves：With Esidilution gradient dilution steps（2）Rat
The first chains of liver organization cDNA, are diluted to 10 respectively-1、10-2、10-3、10-4Times concentration, with the first chain of original cDNA and dilution after
CDNA be template, carry out real time fluorescent quantitative detection, with step（1）In XDH/XO F and XDH/XO R and GAPDH F
It is specific primer with GAPDH R, respectively under following PCR amplification system：25 μ L systems, SYBR Premix Ex Taq II
(Tli RNaseH Plus) 12.5 μ L, positive, each 1 μ L of reverse primer, 1 μ L of cDNA templates, 9.5 μ L of deionized water, are carried out down
Row real-time fluorescence quantitative PCR is expanded：94 DEG C of denaturations 30s, 94 DEG C of degeneration 5s, 59 DEG C of annealing 30s, 40 circulations, 59 DEG C to 94
DEG C per 5s gather first order fluorescence, obtain the solubility curve and standard curve of rat GAPDH genes and XDH/XO genes；
（4）The total serum IgE that rat liver is extracted collects fluorescence and step through real-time fluorescence quantitative PCR detection（3）Gained dissolves peak mark
Directrix curve compares, that is, obtain Wistar rats xanthine dehydrogenase/lysyloxidase gene transcriptional level.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108330173A (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 macaque ABCG2 gene transcription levels |
| CN108330175A (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 macaque SLC22A11/OAT4 gene transcription levels |
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| CN104164481A (en) * | 2014-05-09 | 2014-11-26 | 中国医学科学院医学生物学研究所 | Method for detecting transcription level of tree shrew xanthine dehydrogenase/oxidase gene through RT-PCR |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108330173A (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 macaque ABCG2 gene transcription levels |
| CN108330175A (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 macaque SLC22A11/OAT4 gene transcription levels |
| CN108330173B (en) * | 2017-09-29 | 2022-04-15 | 中国医学科学院医学生物学研究所 | Method for detecting transcription level of ABCG2 gene of macaque by RT-qPCR |
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Application publication date: 20170315 |