CN109609661A - A kind of combination of kidney-yang deficiency exogenous disease mouse model lung tissue qPCR reference gene and its screening technique - Google Patents
A kind of combination of kidney-yang deficiency exogenous disease mouse model lung tissue qPCR reference gene and its screening technique Download PDFInfo
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Abstract
The present invention relates to Protocols in Molecular Biology, in particular to a kind of reference gene combination and its screening technique based on qPCR technology.A kind of screening technique of kidney-yang deficiency exogenous disease mouse model lung tissue qPCR reference gene combination, design 15 reference gene Actb, β 2m, Gapdh, Gusb, Tuba, Grcc10, Eif4h, Rnf187, Nedd8, Ywhea, 18S rRNA, RPL13, Ubc, RPL32, the specific primer of Ppia, upstream and downstream primer sequence is successively as shown in SEQ ID NO.3-32, then using kidney-yang deficiency exogenous disease mouse model cDNA as template, it is utilized respectively the specific primer and RT-qPCR analysis is carried out to 15 reference genes, in conjunction with the absolute value of average value compares between t inspection and group between group, in the convenient and efficient establishment kidney-yang deficiency exogenous disease mouse model lung tissue qPCR of energy The ginseng assortment of genes is Grcc10 and Ppia.Present invention firstly discovers that being combined into Grcc10 and Ppia for kidney-yang deficiency exogenous disease most suitable qPCR reference gene group.It lays the foundation for the subsequent development list of target genes expression study on the disease binding model.
Description
Technical field
The present invention relates to Protocols in Molecular Biology, in particular to a kind of reference gene combination and its sieve based on qPCR technology
Choosing method.
Background technique
Real-Time Fluorescent Quantitative PCR Technique (quantitative real-time PCR, qPCR) is a kind of real-time monitoring
Pcr amplification product and the quantitative approach parsed, have sensibility it is high, it is reproducible, detection speed fastly, high degree of automation
Many advantages, such as, it is widely applied by domestic and foreign scholars at present.The technology is generally required with the stable reference gene of expression to target
The expression of gene is corrected and standardizes.Ideal reference gene should under a variety of experimental conditions, and various types of groups
Knit with expression constant in cell, and its expression quantity be it is approximate, there was no significant difference.However, different types of tissue, no
Reference gene transcriptional level may change under the conditions of the same stage of development and different disposal, therefore, select in suitable
Ginseng gene determines to reduce the difference between detection sample and utilizes the reliable of quantitative fluorescent PCR analysis gene expression dose result
Property.
Previous research is mostly with the numerous reference gene of expression quantity such as rRNA 18S rRNA or 28S rRNA, 3- phosphorus
Acid glycerol dehydrogenase and actin etc. are used as internal reference.However, a large amount of studies have shown that these common reference genes are not
It is also changed with the expression under experiment condition.There are severe deviations in the result that the use of unstable reference gene will lead to qPCR.
Much studies have shown that an extensive general reference gene is not present, researcher needs in each experiment or species
The suitable gene for stablizing expression is found as internal reference.In Bestkeeper, geNorm and NormFinder etc. are widely used as
Join genescreen software, researcher can be helped to select stable reference gene, but these software screening methods in different research
To single internal reference or double reference genes be unsuitable for the reference gene for kidney-yang deficiency exogenous disease lung tissue.In recent years, document
It is more common in yulan[8], rhizoma alismatis[9], teasel[10]The report of the RT-qPCR reference gene screening of equal medicinal plants is more and more, but
It there is no the report about the screening of kidney-yang deficiency exogenous disease mouse model lung tissue RT-qPCR reference gene at present.
Summary of the invention
Goal of the invention of the invention is the t method of inspection between the group for introducing statistical analysis, can efficiently and accurately screen conjunction
Suitable double reference genes combination can be used for research of the subsequent development to kidney-yang deficiency exogenous disease lung tissue destination gene expression.
For achieving the above object, using following technical scheme:
A kind of kidney-yang deficiency exogenous disease mouse model lung tissue qPCR reference gene combination, which is characterized in that the reference gene combination
For Grcc10 and Ppia.
The screening technique of above-mentioned kidney-yang deficiency exogenous disease mouse model lung tissue qPCR reference gene combination, separately designs first
15 reference gene Actb, β 2m, Gapdh, Gusb, Tuba, Grcc10, Eif4h, Rnf187, Nedd8, Ywhea, 18S
The specific primer of rRNA, RPL13, Ubc, RPL32, Ppia, upstream and downstream primer sequence successively as shown in SEQ ID NO.3-32,
Then with kidney-yang deficiency exogenous disease mouse model, cDNA is template, is utilized respectively the specific primer and carries out to 15 reference genes
The kidney-yang deficiency exogenous disease mouse model lung tissue qPCR reference gene combination is established in RT-qPCR analysis.
Preferably, screening technique the following steps are included:
(1) kidney-yang deficiency model is replicated；
(2) kidney-yang deficiency exogenous disease model is established；
(3) RT- PCR detects the expression of the mRNA of H1N1 in lung tissue；
(4) total RNA from animal tissues is extracted；
(5) reverse transcription synthesizes cDNA；
(6) design amplimer carries out RT- PCR amplification；15 reference genes Actb, β 2m, Gapdh, Gusb are separately designed,
Tuba, Grcc10, Eif4h, Rnf187, Nedd8, Ywhea, 18S rRNA, RPL13, Ubc, RPL32, Ppia, the internal reference base
The specific primer sequence of cause is successively as shown in SEQ ID NO.1-30；
(7) the RT-PCR analysis of reference gene；Using the method for dual-gene combination of two, and to the dual-gene just of combination of two
Gene expression difference between normal group and model group carries out T inspection, selects combined P value to be greater than 0.05 or more, and carried out this
For the normal group combined a bit compared with the absolute value of model group difference, the smallest combination of absolute value for screening difference refers to base as double
The optimal combination of cause, analyze Grcc10+Ppia combination gene be kidney-yang deficiency exogenous disease mouse model stabilization reference gene.
Preferably, the method that kidney-yang deficiency model is replicated described in step (1) is that 2mg/mL benzene first is injected intraperitoneally with 4mL/kg
Sour estradiol dilution, one time a day, continuous 7d replicates kidney-yang deficiency model.
Preferably, the method that kidney-yang deficiency exogenous disease model is established described in step (1) is to give postanesthetic mice with kidney-yang deficiency
Collunarium is inoculated with 20 μ L/ of influenza virus H1N1 chick embryo allantoic liquid only, normal to organize the isometric physiological saline of collunarium, virus inoculation
Mouse isolation is fed, and ad lib 6 d of water inlet, dissection is put to death；Wherein the tissue culture infective dose of chick embryo allantoic liquid is
TCID50 2.34×108。
Preferably, RT-PCR amplification program described in step (6) are as follows: 1 circulation, 95 DEG C of 10s are carried out under 95 DEG C of 15min
40 circulations are carried out with 60 DEG C of 32s.
Preferably, the specific steps of the RT-PCR analysis of reference gene described in step (8) are as follows: (1) 15 reference gene
Primer PCR amplification；The expression of (2) 15 reference genes determines；(3) BestKeeper, geNorm and NormFinder are soft
Part is to 15 reference gene stability expression analysis of kidney-yang deficiency exogenous disease model；(4) 15 reference gene combination of two T are examined；(5)
The verifying of reference gene stability；(6) the stabilization internal reference base that Grcc10+Ppia combination gene is kidney-yang deficiency exogenous disease mouse model is obtained
Cause.
Be preferably, TLR3, TLR4 for target gene selected by the verifying of reference gene stability described in step (5),
TLE7,RIG-1；The primer sequence of target gene is successively as shown in SEQ ID NO.31-38.
The present invention refers to gene and double reference assortments of genes in the expression of target gene in the list that software is calculated
It is upper with we filtered out it is double there is different with reference to genes, or even occur on the contrary as a result, therefore BestKeeper,
The optimal reference gene and double genes that refer to that the softwares such as geNorm and NormFinder calculate can not be used as most stable of reference
Gene.This research examines the dual-gene combination for filtering out P > 0.05 in double screening processes with reference to gene by T first,
On the basis of this, the absolute value with model group difference is further normally organized relatively after combination of two, is made with the smallest absolute value of difference
For the optimum combination of reference gene two-by-two.Show the importance of reference gene selection by the verifying of target gene.
This research has selected 15 common reference genes: gene enrich cluster C10 gene (gene rich cluster,
C10 gene, Grcc10), beta-actin (beta-actin, Actb), glycerol-3-phosphate
(glyceraldehyde-3-phosphate dehydrogenase, gapdh), eukaryotic translation initiation factor 4H
The expression development of (eukaryotic translation initiation factor 4H, Eif4h), neural precursor is lowered
Gene 8 (neural precursor cell expressed developmentally down-regulated gene 8,
Nedd8), ring finger protein 187 (ring finger protein 187, Rnf187), tyrosine 3-monooxygenase/tryptophan
5- monooxygenase activator protein ε peptide (tyrosine3-monooxygenase/tryptophan 5-monooxygenase
Activation protein, epsilon, Ywhae), beta-glucuronic acid (beta-glucuronidase, Gusb),
Alpha-tubulin (alpha-tubulin, Tuba), beta-2 microglobulin (beta-2 microglobulin, β 2m), 18S core
Sugared body RNA (18S ribosomal RNA, 18SrRNA), ribosomal protein L 13 (Ribosomal protein L-13,
RPL13), ubiquitin C (Ubiquitin C, Ubc), ribosomal protein L 32 (Ribosomal protein L32, RPL32),
Peptide acyl prolyl isomerase A (peptide acyl preserved ammonia acyl isomerase A, Ppia).It sees
Expression variation of these genes during kidney-yang deficiency exogenous disease (influenza virus H1N1) is examined, screening is suitable for the stabilization in this stage
The reference gene of expression.
Beneficial effect
Present invention firstly discovers that being combined into Grcc10 and Ppia for kidney-yang deficiency exogenous disease most suitable qPCR reference gene group.
Invention introduces the t methods of inspection between the group of statistical analysis, carry out combination of two to 15 kinds of candidate reference genes,
And group difference statistics is carried out to double expressions with reference to gene after combination, it is suitable efficiently and accurately to screen
Double reference gene combinations finally obtain Grcc10+Ppia combination gene in the normal group of difference minimum between model group, can be used as
The stabilization reference gene of kidney-yang deficiency exogenous disease mouse model is the subsequent development list of target genes table on the disease binding model
It lays the foundation up to research.
Detailed description of the invention
Fig. 1 is normal group and model group body weight anus temperature, organ index and virus load；
Fig. 2 is 15 expressions with reference to gene in kidney-yang deficiency exogenous disease model；
Fig. 3 is BestKeeper, geNorm and NormFinder to 15 in the kidney-yang deficiency exogenous disease model stability with reference to gene
Expression analysis；
Fig. 4 is the relative expression quantity of target gene TLR3, TLR4, TLE7, RIG-1.
Specific embodiment
The principle of the invention and feature are described with reference to the accompanying drawing, illustrated embodiment is served only for explaining the present invention, and
Non-limiting the scope of the present invention.
Embodiment 1
1 materials and methods
1.1 animal
SPF grades of Balb/c male mices, body weight 18-20 g, purchase please experimental animal from Jinan friend and breed Co., Ltd, license
Card number: the Shandong SCXK() 20140007.
1.2 reagent
Oestradiol benzoate (lot number: 140627, be purchased from the second hormone of Ningbo factory, 2 mg/mL), H1N1 influenza virus (Beijing
Square strain is quoted from the Chinese institute of viruses CDC), 0.9% sodium chloride injection (Cisen Pharmaceutical Co., Ltd., lot number:
1601042161), RNAstore sample saves liquid-DP408, total RNA from animal tissues extracts kit-DP431(RNAprep
Pure Tissue Kit), cDNA reverse transcription reagent box-KR106, SYBR Green PCR kit for fluorescence quantitative-FP205(days
Root company).
1.3 instrument
Quawell ultramicron ultraviolet specrophotometer (USA), fluorescence quantitative PCR instrument (CFX Connect Real-Time
System, Bole), anus temperature tester: BAT-12 miniature probe thermometer (U.S. Physitemp Instruments, BAT-12),
Toy Anesthesia machine (MIDMARK company, the U.S., model: VMR).
1.4 primer
15 candidate reference genes have been selected in this experiment.Candidate gene is Actb, β 2m, Gapdh, Gusb, Tuba, Grcc10,
Eif4h, Rnf187, Nedd8, Ywhea, 18S rRNA, RPL13, Ubc, RPL32, Ppia.Use NCBI BLAST(http: //
Blast.ncbi.nlm.nih.gov/Blast.cgi) the nucleotide sequence design primer identified, upstream and downstream primer sequence is successively
As shown in SEQ ID NO.3-32；Primer is purchased from Beijing six directions Hua Da Technology Co., Ltd., is shown in Table 1.
2 experimental methods
2.1 animal packet
20 Balb/c mouse are randomly divided into 2 groups according to weight, anus temperature, normal group, model group.
2.2 modeling
2.2.1 kidney-yang deficiency model is replicated
The identical nature and flavor difference channel tropism Chinese medicine of bibliography causes the method in the influence of mice with kidney-yang deficiency with 4mL/kg abdomen to estradiol
Chamber injects 2mg/mL oestradiol benzoate dilution, and one time a day, continuous 7d replicates kidney-yang deficiency model, measures autonomic activities, anus
Temperature, swimming time, with this decision model success or not.
2.2.2 kidney-yang deficiency exogenous disease model is established
Reference literature kidney-yang deficiency exogenous disease Establishment of mouse model and method in Ephedra asarum monkshood soup intervention study simultaneously change virus
Collunarium dosage, give postanesthetic mice with kidney-yang deficiency collunarium inoculation influenza virus H1N1 chick embryo allantoic liquid (TCID50 2.34 ×
108) 20 μ L/ only, normal to organize the isometric physiological saline of collunarium, the mouse of virus inoculation, which is isolated, to be fed, ad lib water inlet 6
D, dissection are put to death.
2.3 index determining
2.3.1 each group mouse weight, anus temperature detector survey weight, the anus temperature of daily measurement mouse.
2.3.2 organ index
Lung tissue, heart, liver are taken after dissection, kidney, for calculating organ index, is weighed with weighing after normal saline flushing
Tissue samples afterwards immerse RNAstore with 1:10 ratio.
2.4 Real Time PCR detect the expression of the mRNA of H1N1 in lung tissue
2.4.1 Total RNAs extraction
10-20 mg tissue is weighed, extracts its total serum IgE by total RNA from animal tissues extracts kit method.Pass through Quawell ultra micro
The yield and purity for measuring ultraviolet specrophotometer measurement RNA use the sample of dulling luminosity ratio OD 260/280 between 1.8 and 2.1
In further analysis.
2.4.2 reverse transcription synthesizes cDNA.
According to cDNA Reverse Transcription cassette method, using 20 42 DEG C of μ L Transcription Systems, 3min removes contaminating genomic DNA, will
Total serum IgE and reverse transcriptase FastQuant RT Enzyme, 42 DEG C, 15min synthesizes the first chain cDNA.CDNA is stored in -20 DEG C
It is spare.
2.4.3 Real-time PCR
For each reaction, 20 μ L mixtures contain 2 μ L cDNA, and forward and reverse primer sequence is successively such as SEQ ID NO.1-
Shown in 30, each 6 pmol, 10 μ L 2 × SuperReal PreMix Plus(Green containing SYBR I).Amplification program is as follows:
1 circulation is carried out under 95 DEG C of 15min, 95 DEG C of 10s and 60 DEG C of 32s carry out 40 circulations.After amplification, heat denatured is obtained
The melting curve of PCR product is to verify specific amplification.
Using standard curve calculate each primer pair exponential phase qPCR efficiency (cDNA of reverse transcription carry out 4 times it is continuous dilute
Release), average threshold circulation (Ct) value of each serial dilution maps to logarithm.According to equation E=10(-1/slope)
× 100 calculate cDNA dilution gfactor, and wherein slope is the gradient of linear regression line.
2.4.4 H1N1 Viral Quantification
H1N1 influenza virus M gene is detected using the Real-Time PCR of SYBR Green dye method.H1N1 influenza virus overall length
M gene standard curve use based on primer (F:5 '-CTGAGAAGCAGATACTGGGC-3 ', R:5 '-
CTGCATTGTCTCCGAAGAAAT-3 ') Real-Time PCR amplification.PCR product is cloned into pTarget plasmid, uses
MEGAshortscript kit is for being transcribed in vitro.The absolute copy number of H1N1 influenza virus M gene passes through standard curve meter
It calculates.
2.5 verifying primers
Based on being used in conjunction with as endogenous control gene in previous research, 15 candidate reference genes, candidate base have been selected
Because being Actb, β 2m, Gapdh, Gusb, Tuba, Grcc10, Eif4h, Rnf187, Nedd8, Ywhea, 18S rRNA respectively,
RPL13, Ubc, RPL32 and Ppia.
It is as follows for verifying the selected primer with reference to gene: TLR3-F:CAGGATACTTCTCGCCTT and TLR3-R:
TGGCCGCTGAGTTTGTTTTC；TLR4-F:CATGGATCAGAAACTCAGCAAAGTC and TLR4-R:
CATGCCATGCCTTGTCTTCA；TLR7-F:CTGGAGTTCAGCAACCATT and TLR7-R:
GTTATCACCGCTCCGCTCCATAGAA；RIG-1-F:GCAGGGGGGGGGGGTTACTGTGGACTTTGTG and RIG-1-R:
TGCCATCTCTCCTTTGTGTCT。
The analysis of 2.7 data
These data are used to assess ginseng by three common statistics programs (i.e. geNorm, NormFinder and BestKeeper)
Examine the stability of gene.According to formula: 2−ΔCtCt value is converted to nonstandardized technique relative quantity by (Ct=corresponding Ct value-minimum Ct).
The relative quantity that the calculating of geNorm and NormFinder is converted based on these；Original Ct value is directly divided by BestKeeper software
Analysis.
2.8 statistical analysis
T is examined carrying out group to experimental data using 22.0 software of SPSS, *P< 0.05 and * * P< 0.01 thinks there is system
Meter learns meaning.
3 results
3.1 weight, anus temperature, organ index and virus load
Normal group mouse fur is dry and comfortable smooth, and the state of mind is good, and active active, weight is cumulative.Model group mouse infection
H1N1 virus for 24 hours after, symptom initial stage be torpescence, scratch nose, apathetic, the symptoms such as hunchbacked atrophy, weight with normal group
Mouse, which is compared, significant difference, and anus temperature is significantly lower than normal group mouse (Fig. 1 a, b).Model Lung Exponent significantly increases, seminal vesicle
Gland index is substantially reduced (Fig. 1 c), while the absolute copy number of M gene 6.1 of the influenza virus H1N1 of the lung tissue of model mice ×
106/ ml or more shows kidney-yang deficiency exogenous disease model construction success (Fig. 1 d).
3.2 refer to gene primer PCR amplification efficiency and specificity
Production 15 standard curves with reference to gene after qRT-PCR reaction, the results show that each coefficient R20.990 is all larger than,
15 pairs of amplification efficiencies referring to gene (table 1) between 91%-98.9%.The above results show respectively with reference to gene primer specificity
Meet qRT-PCR requirement, can be used for the estimation of stability with reference to gene.
1 qPCR primer of table and gene sequence information
The expression of 3.3 candidate reference genes
Ct value of 15 candidate reference genes in kidney-yang deficiency exogenous disease model mice is between 13.861-23.572, average value
It is 18.591.Wherein Actb expression highest, Ct value is minimum, is 13.861.The expression of Gusb is minimum, and Ct value is maximum,
For 23.572(Fig. 2 a).Relative quantification is carried out after the normalized processing of Ct value of 15 candidate genes, uses t test statistics respectively
Analyze each candidate reference gene in the normal group of significant difference between model group.The result shows that in this 15 candidates
Joining gene relative expression quantity all has significant difference (P < 0.05, or P < 0.01) (Fig. 2 b) between model group at normal group.
Stability of 3.4 BestKeeper, geNorm and NormFinder softwares to kidney-yang deficiency exogenous disease model reference gene
Expression analysis
As seen from the figure, the difference that these three common reference gene screening softwares are obtained is expressed not with reference to the stability of gene
Unanimously, it can find that the stability of Nedd8 gene is best, and the stability of β 2m gene is worst through the analysis of BestKeeper software；
GeNorm software analysis finds that the stability of Ywhea and Tuba gene is best, and the stability of β 2m gene is worst；
NormFinder software analysis finds that the stability of 18SrRNA gene is best, and the stability of β 2m gene is worst (Fig. 3).Separately
Outside, optimal double reference genes that NormFinder software is analyzed are Eif4h+RPL32(table 2).
2 Normfinder calculated result of table
3.5 examine with reference to gene combination of two T
Using the method for dual-gene combination of two, and to the dual-gene gene table at normal group between model group of combination of two
T inspection is carried out up to difference, has the P value of 15 kinds of combinations to be greater than 0.05 or more, does not have the discrepant assortment of genes two-by-two again between these groups
The normal group of each combination is carried out compared with the absolute value of model group difference, it is believed that with the smallest group of the absolute value of difference
It closes and can be used as double optimal combinations with reference to gene.Experimental result shows that Grcc10+Ppia two refers to the difference of the assortment of genes
Minimum (0.009), therefore Grcc10+Ppia combination can be used as the optimal selection (table of kidney-yang deficiency exogenous disease lung tissue sample internal reference
3, table 4).
The T of 3 15 kinds of reference gene combination of two of table examines (P value)
15 kinds of reference gene combination of two T that table 4 filters out examine the difference of P value and average value
3.6 verify with reference to gene stability
In order to verify double stability and reliability with reference to gene, we have selected 4 verifying genes to analyze the phase of target gene
To expression: toll sample receptor 3 (TLR3), toll sample receptor 4 (TLR4), toll sample receptor 7 (TLR7) and cytosol view
Yellow acid induced gene -1 (RIG-1).As a result, it has been found that selecting double with reference to gene Grcc10+Ppia and Eif4h+RPL32 and not
Gene (Nedd8, Ywhea, 18s rRNA) is referred in target gene TLR3, TLR7, RIG-1 with stable list is showed in software
Occur certain difference on expression, and in the expression of TLR4 gene, Eif4h+RPL32 combined reference gene and
There is opposite result on expression in 18s rRNA (see Fig. 4).
In molecular biology research, gene expression analysis is one of research most common strategy of gene.QRT-PCR because
Its expression that Relative gene is widely used in accuracy.In this technique, select to stablize the gene expressed as reference
Gene is vital.Cell, tissue or other stress handle after can be expressed with constant level, and have and target
The comparable expression of gene.The study find that it is different using the optimal reference gene that different software for calculation obtain, and
In kidney-yang deficiency exogenous disease mouse model, using a kind of expression that can not accurately determine target gene with reference to gene, therefore to 15 kinds
Candidate reference gene carries out combination of two, and carries out group difference system to double expressions with reference to gene after combination
Meter finally obtains Grcc10+Ppia combination gene in the normal group of difference minimum between model group.By verifying target gene
TLR3, TLR4, TLR7 and RIG-1 show that the list that software is calculated refers to gene and double reference assortments of genes in target gene
Expression on we filtered out it is double there is different with reference to genes, or even occur opposite as a result, therefore
The optimal reference gene and double reference genes that the softwares such as BestKeeper, geNorm and NormFinder calculate can not be as most
Stable reference gene.This research filters out the biradical of P > 0.05 by T inspection first in double screening processes with reference to gene
Because of combination, on this basis, the absolute value with model group difference is further normally organized relatively after combination of two, it is the smallest with difference
Optimum combination of the absolute value as reference gene two-by-two.Show the importance of reference gene selection by the verifying of target gene.
The study find that Grcc10+Ppia combination gene can be used as the stabilization reference gene of kidney-yang deficiency exogenous disease mouse model, be subsequent
Development list of target genes expression study on the disease binding model lays the foundation.
Sequence table
<110>Shandong Traditional Chinese Medicine University
<120>a kind of kidney-yang deficiency exogenous disease mouse model lung tissue qPCR reference gene combination and its screening technique
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<212>DNA
<213>artificial sequence
TGGGAAGGAG ATTGAGATAG 20
<210>18
<211>19
<212>DNA
<213>artificial sequence
TTGCTTGCCA CTGTAGATG 19
<210>19
<211>18
<212>DNA
<213>artificial sequence
CCCATTCGTT TAGGTCTT 18
<210>20
<211>18
<212>DNA
<213>artificial sequence
TCCACAGCGT CAGGTTAT 18
<210>21
<211>21
<212>DNA
<213>artificial sequence
TTGACGGAAG GGCACCACCA G 21
<210>22
<211>21
<212>DNA
<213>artificial sequence
GCACCACCAC CCACGGAATC G 21
<210>23
<211>20
<212>DNA
<213>artificial sequence
GTACGCTGTG AAGGCATCAA 20
<210>24
<211>20
<212>DNA
<213>artificial sequence
ATCCCATCCA ACACCTTGAG 20
<210>25
<211>20
<212>DNA
<213>artificial sequence
GCCCAGTGTT ACCACCAAGA 20
<210>26
<211>20
<212>DNA
<213>artificial sequence
CCCATCACAC CCAAGAACA 19
<210>27
<211>17
<212>DNA
<213>artificial sequence
GAACTGGCGG AAACCCA 17
<210>28
<211>20
<212>DNA
<213>artificial sequence
GGATCTGGCC CTTGAACCTT 20
<210>29
<211>20
<212>DNA
<213>artificial sequence
CGCTTGCTGC AGCCATGGTC 20
<210>30
<211>20
<212>DNA
<213>artificial sequence
CAGCTCGAAG GAGACGCGGC 20
<210>31
<211>18
<212>DNA
<213>artificial sequence
CAGGATACTT CTCGCCTT 18
<210>32
<211>20
<212>DNA
<213>artificial sequence
TGGCCGCTGA GTTTGTTTTC 20
<210>33
<211>25
<212>DNA
<213>artificial sequence
CATGGATCAG AAACTCAGCA AAGTC 25
<210>34
<211>20
<212>DNA
<213>artificial sequence
CATGCCATGC CTTGTCTTCA 20
<210>35
<211>19
<212>DNA
<213>artificial sequence
CTGGAGTTCA GCAACCATT 19
<210>36
<211>25
<212>DNA
<213>artificial sequence
GTTATCACCG CTCCGCTCCA TAGAA 25
<210>37
<211>31
<212>DNA
<213>artificial sequence
GCAGGGGGGG GGGGTTACTG TGGACTTTGT G 31
<210>38
<211>19
<212>DNA
<213>artificial sequence
TGCCATCTCT CCTTTGTGTC T 21
Claims (8)
1. a kind of kidney-yang deficiency exogenous disease mouse model lung tissue qPCR reference gene combination, which is characterized in that the reference gene group
It is combined into Grcc10 and Ppia.
2. a kind of screening technique of kidney-yang deficiency exogenous disease mouse model lung tissue qPCR reference gene combination described in claim 1,
It is characterized in that, 15 reference genes Actb, β 2m, Gapdh, Gusb, Tuba, Grcc10, Eif4h are separately designed first,
The specific primer of Rnf187, Nedd8, Ywhea, 18S rRNA, RPL13, Ubc, RPL32, Ppia, upstream and downstream primer sequence according to
Secondary, then with kidney-yang deficiency exogenous disease mouse model, cDNA is template as shown in SEQ ID NO.1-30, is utilized respectively the specificity
15 reference genes of primer pair carry out RT-qPCR analysis, establish the kidney-yang deficiency exogenous disease mouse model lung tissue qPCR internal reference base
Because of combination.
3. screening technique according to claim 2, which comprises the following steps:
(1) kidney-yang deficiency model is replicated；
(2) kidney-yang deficiency exogenous disease model is established；
(3) RT- PCR detects the expression of the mRNA of H1N1 in lung tissue；
(4) total RNA from animal tissues is extracted；
(5) reverse transcription synthesizes cDNA；
(6) design amplimer carries out RT- PCR amplification；15 reference genes Actb, β 2m, Gapdh, Gusb are separately designed,
Tuba, Grcc10, Eif4h, Rnf187, Nedd8, Ywhea, 18S rRNA, RPL13, Ubc, RPL32, Ppia, the internal reference base
The specific primer sequence of cause is successively as shown in SEQ ID NO.1-30；
(7) the RT-PCR analysis of reference gene；Using the method for dual-gene combination of two, and to the dual-gene just of combination of two
Gene expression difference between normal group and model group carries out T inspection, selects combined P value to be greater than 0.05 or more, and carried out this
For the normal group combined a bit compared with the absolute value of model group difference, the smallest combination of absolute value for screening difference can be used as double references
The optimal combination of gene, analyze Grcc10+Ppia combination gene be kidney-yang deficiency exogenous disease mouse model stabilization reference gene.
4. screening technique according to claim 3, which is characterized in that replicate the side of kidney-yang deficiency model described in step (1)
Method is that 2mg/mL oestradiol benzoate dilution is injected intraperitoneally with 4mL/kg, and one time a day, continuous 7d replicates kidney-yang deficiency model.
5. screening technique according to claim 3, which is characterized in that establish kidney-yang deficiency exogenous disease model described in step (1)
Method be to give postanesthetic mice with kidney-yang deficiency collunarium to be inoculated with 20 μ L/ of influenza virus H1N1 chick embryo allantoic liquid only, normal group drop
The isometric physiological saline of nose, the mouse of virus inoculation, which is isolated, to be fed, and ad lib 6 d of water inlet, dissection is put to death；Wherein chicken embryo is urinated
The tissue culture infective dose of cyst fluid is TCID50 2.34 × 108。
6. screening technique according to claim 3, which is characterized in that RT- PCR amplification program described in step (6) are as follows:
1 circulation is carried out under 95 DEG C of 15min, 95 DEG C of 10s and 60 DEG C of 32s carry out 40 circulations.
7. according to screening technique as claimed in claim 3, which is characterized in that the RT-PCR of reference gene described in step (8) is analyzed
Specific steps are as follows: the amplification of (1) 15 reference gene primer PCR；The expression of (2) 15 reference genes determines；(3)
BestKeeper, geNorm and NormFinder software are to 15 reference gene stability expression analysis of kidney-yang deficiency exogenous disease model；
(4) 15 reference gene combination of two T are examined；(5) reference gene stability is verified；(6) obtaining Grcc10+Ppia combination gene is
The stabilization reference gene of kidney-yang deficiency exogenous disease mouse model.
8. screening technique according to claim 7, which is characterized in that be used for reference gene stability described in step (5)
The selected target gene of verifying is TLR3, TLR4, TLE7, RIG-1；The primer sequence of target gene is successively such as SEQ ID
Shown in NO.31-38.
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