CN105779624A - Primer combination for detecting expression level of human-derived CHOP gene and technical field of application of primer combination - Google Patents

Primer combination for detecting expression level of human-derived CHOP gene and technical field of application of primer combination Download PDF

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CN105779624A
CN105779624A CN201610252885.3A CN201610252885A CN105779624A CN 105779624 A CN105779624 A CN 105779624A CN 201610252885 A CN201610252885 A CN 201610252885A CN 105779624 A CN105779624 A CN 105779624A
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chop
gene
expression level
primer
primer combination
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蒋明
于丽华
郭佳
刘敏亚
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SUZHOU RESEARCH INSTITUTE OF TONGJI UNIVERSITY
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Abstract

The invention belongs to the technical field of genes, and relates to a primer combination for detecting the expression level of a human-derived CHOP gene. A specific primer of the human-derived CHOP gene and a specific primer of a ribosomal protein RPL19 gene are designed to detect the expression level of the CHOP gene. The specific primer combination and a corresponding method can be used for specifically detecting the expression level of the human-derived CHOP gene to avoid interference of homologous genes, and takes RPL19 which cannot be affected by endoplasmic reticulum stress as an internal reference to accurately quantify the expression level of the CHOP gene. The detection system adopts an SYBR green fluorochrome method, so that no probe is used, and reagents are convenient to use; only relevant detection templates are needed; complicated operating steps are not required, and the detection process can be completed within 1.2 h.

Description

A kind of combination of primers for detecting humanized's CHOP gene expression dose and applied technical field thereof
Present disclosure belongs to gene technology field, the present invention relates to a kind of combination of primers for detecting humanized's CHOP gene expression dose.
Background technology CHOP, also referred to as GADD153, is found in DNA damage reaction at first.CHOP is wide expression in mammalian cell, is the idiosyncratic transcription factor of er stress, belongs to C/EBP transcription factor family.Under normal physiological conditions, CHOP is expressed in eukaryotic cytoplasm on a small quantity, when cell generation stress, calcium is unbalance etc. time, CHOP assembles in a large number and is expressed in eukaryotic cell nucleus, active cell apoptotic signal path, by affecting Bcl-2 anti-apoptotic proteins and expressing, interference cell oxidation reaction or affect the activity etc. of Caspase-12 and promote apoptosis.CHOP is the important albumen of er stress inducing cell apoptosis, and it can suppress Wnt/TCF signal path by N-terminal transcriptional activation domain, and tumor invasion, differentiation of stem cells and body development are had potential impact.There are some researches show that hypoxic-ischemic damage can cause vascular endothelial cell generation er stress and inflammatory reaction, by raising the expression of CHOP, it is suppressed that the proliferation activity of cell, promote apoptosis.Also studies have found that CHOP high expressed in human glioma, in normal cerebral tissue, expression is relatively low, and prompting exists er stress in glioma, promotes abnormal proliferative cell generation apoptosis.In addition, big quantity research shows, using CHOP as the antitumor drug of target spot development of new, there is certain feasibility, CHOP is activated by AP1 and the AARE1 region that can pass through to activate in CHOP promoter, promote its transcriptional expression, thus causing the expression of signal protein Bcl2 downstream to decline, causing death of neoplastic cells, reaching antineoplastic purpose.Therefore by the research of CHOP expression being contributed to the pathogenesis of clear and definite disease and the Outcome measure to medicine.
The research of CHOP expression is studied from protein level except the method such as SABC and Western immunoblotting of employing, it is possible to from the expression of mRNA level in-site research CHOP, including sxemiquantitative reverse transcriptional PCR and fluorescence quantifying PCR method etc..Generally operate comparatively laborious with albumen for detection order calibration method, consuming time.Sxemiquantitative reverse transcriptional PCR needs electrophoresis detection, and accuracy is not high enough.Fluorescence quantifying PCR method is a kind of high sensitivity, simple and efficient detection method, simple to operate, the detection of SYBRgreen dye method can be applied, without using fluorescent probe, design is simple, adopting quantitative fluorescent PCR relative quantitation method by comparing, with the metastable house-keeping gene of expression, the expression reflecting genes of interest, can well assist the research of CHOP expression, whole PCR course of reaction only needs to complete for 1.2 hours.Cell house keeping gene need to select to maintain the genoid that cell bottom line function is indispensable, be intended to expression in all cells, and expression is less by such environmental effects.There are some researches show that the more commonly used actin house-keeping gene is affected by er stress, expression can reduce, and is not suitable for the reference gene for er stress.Ribosomal protein gene product is to maintain necessary to cell activities, is expressed in all cells, and it expresses the impact being only limited by initiating sequence or promoter and RNA polymerase interaction, and is not regulated by other mechanism, can be used as the reference gene of er stress.
The specific primer of summary of the invention present invention specific primer and ribosomal protein RPL19 gene by designing humanized's CHOP gene, uses it for the detection of CHOP gene expression dose.The operation principle of the present invention be the method for application quantitative fluorescent PCR SYBRgreen fluorescent dye according to relative quantification principle, namely 2-△△CTMethod determines gene expression dose.
The present invention comprises two set primer systems, and a set of primer is the specific primer for humanized's CHOP gene；Another set of primer is the specific primer of the ribosomal protein RPL19 gene design for people.
CHOP primer sequence is designed in Table 1 according to the humanCHOPmRNA sequence (serial number: NM_001195053.1) that NCBI announces:
Table 1.CHOP design of primers
Primer Sequence
F-chop GGAGCTGGAAGCCTGGTATG
R-chop GCAGGGTCAAGAGTGGTGAA
MRNA sequence (serial number: NM_000981.3) according to the NCBI humanRPL19 announced designs primer sequence in Table 2:
Table 2.RPL19 design of primers
Primer Sequence
F-rpl CGAGCGAGCTCTTTCCTTT
R-rpl AGACCTTCTTCTTGCCACAGC
Two set primers of technique effect present invention design can the mrna expression level of specific detection CHOP and RPL19 gene, without non-specific amplification, selected house-keeping gene expression is not affected by er stress, the expression of CHOP gene can be carried out accurate quantitative analysis.The detection system of the present invention adopts SYBRgreen fluorescent dye method, it is not necessary to using probe, reagent is easy to use, only need to add coherent detection template, it is not necessary to loaded down with trivial details operating procedure, and detection only needs 1.2h to complete.
Accompanying drawing explanation
Accompanying drawing 1 is the melt curve analysis of CHOP and RPL19 gene amplification product in the embodiment of the present invention 1, and wherein A figure is CHOP gene amplification product melt curve analysis；B figure is RPL19 gene amplification product melt curve analysis.
Accompanying drawing 2. is the MANF albumen impact on PD cell model CHOP gene expression dose in the embodiment of the present invention 2.
Detailed description of the invention
Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described in further detail.Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.
The AcetaqSYBRsupermix adopting promise only to praise in the embodiment of the present invention, adopts 25 microlitre reaction systems, and quantitative real time PCR Instrument adopts ABI7500.
When utilizing specific primer sets and method detection humanized's CHOP gene expression dose of the present invention, basic operation is:
Step 1. cell RNA extracts
Adopting commercial kit to extract cell RNA, ultraviolet spectrophotometer measures RNA concentration, and controls to extract quality OD260/280～2.0.The embodiment of the present invention adopts sky root total RNA extraction reagent box to extract cell RNA, and concrete operations by specification carries out；
Step 2.cDNA reverse transcription
Taking about 500ngRNA adopts commercial kit to carry out cDNA reverse transcription.The present embodiment adopts the commercial kit PrimeScript of TakaraTMRTMasterMix(PerfectRealTime) carrying out reverse transcription, concrete operations by specification carries out；
Step 3. quantitative fluorescent PCR reacts
In experiment, every kind of template is separately added in two kinds of quantitative fluorescent PCR reaction systems and carries out CHOP and RPL19 gene test respectively, and it is as follows that a kind of reaction system comprises component:
1×AcetaqSYBRsupermix
Each 0.1～0.5 μM of forward primer (F-chop)
Each 0.1～0.5 μM of downstream primer (R-chop)
Template (cDNA of 1:5 dilution) 5 μ l
Cumulative volume 25 μ l
Another kind is:
1×AcetaqSYBRsupermix
Each 0.1～0.5 μM of forward primer (F-rpl)
Each 0.1～0.5 μM of downstream primer (R-rpl)
Template (cDNA of 1:5 dilution) 5 μ l
Cumulative volume 25 μ l
Quantitative fluorescent PCR reaction condition: 95 DEG C of 5min, then reruns 40 and circulates (concrete 95 DEG C of 10s, 60 DEG C of 34s), detects fluorescence signal during annealing.Melt curve analysis analysis can be carried out, the specificity of detection primer amplification after end of run；
Step 4. Analysis of test results
The CT value obtaining each reaction is set according to the automatic baseline of system and logarithmic (log) phase threshold value, calculates 2-△△CTValue, the multiple that namely experimental group CHOP mrna expression compares with matched group, △ △ CT=(CTCHOP(experimental group)-CTrpl19(experimental group))-(CTCHOP(matched group)-CTrpl19(matched group)).
In embodiment one slow virus infected cell, CHOP gene expression dose is analyzed
By the packaging slow virus Infection in Vitro 293T cell containing GRP78 gene interference sequence.24 orifice plates are inoculated 293T cell, inoculum density 3*105/ml.Cultivate cell density after 24h and be about 50%-60%, carry out slow virus infection by MOI5:1, and set and be not added with virus negative control.At 37 DEG C, in 5%CO2 incubator, cultivate 24h, change the fresh DMEM in high glucose culture medium containing 10%FBS and continue to cultivate 24h, suck supernatant, PBS three times, collect cell, extract cell RNA.Take 500ngRNA reverse transcription cDNA respectively, adopt the expression of fluorescence quantitative PCR method detection CHOP gene, using RPL19 gene as internal reference, and compare with the cell not adding viral infection.Testing result is in Table 3:
The comparison of table 3. slow virus infected cell and compared with control cells CHOP expression
Table 3, it is shown that slow virus infected cell CHOP gene expression dose is significantly raised compared with cellular control unit, is 11.76 times of cellular control unit, illustrates that cell is had certain toxic action by slow virus infection.All only there is single melt curve analysis peak (see figure 1) in the melt curve analysis of CHOP and RPL19 primer amplification, illustrates that primer specificity is better, without non-specific amplification.
Embodiment 2. studies the protective effect of Manf albumen in parkinson disease cell model by detecting the expression of CHOP gene
Adopting 6-hydroxy dopamine (6-OHDA) to make the protective effect of parkinson disease cell model detection Manf albumen of SH-SY5Y cell injury, experiment is divided into matched group (DMEM) group, 6-OHDA(50 μm of ol/L) group and 6-OHDA(50 μm of ol/L)+Manf(4 μ g/ml) group.Collect cell after adding 6-OHDA effect 6h and 16h after giving Manf albumen 4h in advance respectively, extract RNA, after reverse transcription, adopt quantitative fluorescent PCR to measure the expression of CHOP gene.By comparing with matched group, calculate relative expression levels's (see figure 2) of each group of CHOP, result shows no matter Manf albumen presence or absence, behind 6 hours and 16h, 6-OHDA equal inducing cell CHOP expresses significantly raised, illustrate that 6-OHDA can make cell produce er stress, promote the expression of CHOP gene.Manf albumen does not make the transcriptional level of CHOP gene reduce, and the protective effect of prompting Manf albumen realizes not by the pretranscriptional control to CHOP gene.
By the above embodiments it is known that, the specific primer sets of the present invention and corresponding method, can the expression of specific detection humanized's CHOP gene, avoid the interference of homologous genes, and using the RPL19 that do not affected by er stress as internal reference, improve the detection accuracy of humanized's CHOP gene expression dose, detection process is simple, efficient.
Above specific embodiments of the invention being described in detail, but it is intended only as example, the present invention is not restricted to particular embodiments described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and replacement are also all among scope of the invention.Therefore, the equalization made without departing from the spirit and scope of the invention converts and amendment, all should contain within the scope of the invention.
SEQUENCELISTING
<110>Tongji University Suzhou academy
<120>a kind of combination of primers for detecting humanized's CHOP gene expression dose and application thereof
<130>2016
<160>4
<170>PatentInversion3.5
<210>1
<211>20
<212>DNA
<213>artificial sequence
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<221>misc_feature
<223>pcr amplification primer
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<211>20
<212>DNA
<213>artificial sequence
<220>
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<223>pcr amplification primer
<400>2
gcagggtcaagagtggtgaa20
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<211>19
<212>DNA
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<221>misc_feature
<223>pcr amplification primer
<400>3
cgagcgagctctttccttt19
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<213>artificial sequence
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<400>4
agaccttcttcttgccacagc21

Claims (5)

1., for detecting a specific primer sets for humanized's CHOP gene expression dose, this combination comprises following primer sequence:
The combination of primers of CHOP gene test:
Forward primer F-chop:GGAGCTGGAAGCCTGGTATG
Downstream primer R-chop:GCAGGGTCAAGAGTGGTGAA
The combination of primers of RPL19 reference gene detection:
Forward primer F-rpl:CGAGCGAGCTCTTTCCTTT
Downstream primer R-rpl:AGACCTTCTTCTTGCCACAGC.
2. the method detecting humanized's CHOP gene expression dose, it is characterised in that use specific primer sets as claimed in claim 1 to carry out.
3. the test kit being used for detecting humanized's CHOP gene expression dose, it is characterised in that include specific primer sets as claimed in claim 1.
4. test kit according to claim 3, it is characterised in that also include archaeal dna polymerase, dNTP and buffer.
5. test kit according to claim 4, it is characterised in that described polymerase is thermostable DNA polymerase.
CN201610252885.3A 2016-04-22 2016-04-22 Primer combination for detecting expression level of human-derived CHOP gene and technical field of application of primer combination Pending CN105779624A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1814792A (en) * 2005-02-02 2006-08-09 苏州大学附属第一医院 Real-time quantitative PCR detecting human DDIT3 transcript kit
US20130260458A1 (en) * 2012-03-30 2013-10-03 National University Corporation Chiba University Method of inducing differentiation of human pluripotent stem cell into hepatic progenitor cell
WO2015142713A1 (en) * 2014-03-17 2015-09-24 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Compositions and methods for reducing c/ebp homologous protein activity in myeloid-derived suppressor cells

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1814792A (en) * 2005-02-02 2006-08-09 苏州大学附属第一医院 Real-time quantitative PCR detecting human DDIT3 transcript kit
US20130260458A1 (en) * 2012-03-30 2013-10-03 National University Corporation Chiba University Method of inducing differentiation of human pluripotent stem cell into hepatic progenitor cell
WO2015142713A1 (en) * 2014-03-17 2015-09-24 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Compositions and methods for reducing c/ebp homologous protein activity in myeloid-derived suppressor cells

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BALAJI BALAKRISHNAN等: "Activation of the Cellular Unfolded Protein Response by Recombinant Adeno-Associated Virus Vectors", 《PLOS ONE》 *
BALAKRISHNAN, BALAJI等: "Activation of the Cellular Unfolded Protein Response by Recombinant Adeno-Associated Virus Vectors: Implications in Gene Therapy", 《MOLECULAR THERAPY》 *
未知: "Homo sapiens ribosomal protein L19 (RPL19), mRNA", 《NCBI GENBANK》 *

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