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Primer probe set, kit and method for quantitative detection of expression level of small integral membrane protein 3 (SMIM3) gene

A gene expression and quantitative detection technology, applied in the field of biochemical detection, can solve the problems of unclear function and less research on SMIM3

Pending Publication Date: 2020-05-05
THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

SMIM3 may be involved in the formation or regulation of ion channels, but there are very few studies on SMIM3, and its function is still unclear

Method used

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  • Primer probe set, kit and method for quantitative detection of expression level of small integral membrane protein 3 (SMIM3) gene
  • Primer probe set, kit and method for quantitative detection of expression level of small integral membrane protein 3 (SMIM3) gene
  • Primer probe set, kit and method for quantitative detection of expression level of small integral membrane protein 3 (SMIM3) gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Primer probe set for detection of SMIM3 gene expression according to the present invention

[0028] The primer probe set of the present invention comprises component A and component B, and said component A comprises:

[0029] Upstream primer SMIM3-FP: 5'-TCATCATGACCTCGTTGTTGCT-3 (ie the nucleotide sequence shown in SEQ ID NO.1), downstream primer SMIM3-RP: 5'-CTCAAACAGCCCCACTAAGGAT-3' (ie the nucleic acid sequence shown in SEQ ID NO.2 nucleotide sequence) and probe SMIM3-probe: FAM-ACTGCAGTAATCATCTATCGCATGCGGAC-BHQ (ie, the nucleotide sequence shown in SEQ ID NO.3), wherein the 5' end of the probe SMIM3-probe is labeled with a FAM luminescent group, and the 3' The end is labeled with a fluorescent quencher group BHQ;

[0030] The component B includes:

[0031] Upstream primer ABL1-FP: 5'-TGGAGATAACACTCTAAAGCATAACTAAAGGT-3' (ie the nucleotide sequence shown in SEQ ID NO.4), downstream primer ABL1-RP: 5'-GATGTAGTTGCTTGGGACCCA-3' (ie the nucleotide sequence sho...

Embodiment 2

[0032] Example 2 The kit for detecting the expression level of SMIM3 gene according to the present invention

[0033] The kit of the present invention includes component A and component B in the embodiment, also includes positive control plasmid and internal reference control plasmid, upstream primer SMIM3-FP in component A: downstream primer SMIM3-RP: probe SMIM3- The molar ratio of probe is 3:3:2; In component B, upstream primer ABL1-FP: downstream primer ABL1-RP: the molar ratio of probe ABL1-probe is 3:3:2;

[0034] Wherein the positive control plasmid is a positive plasmid inserted into the SMIM3 gene fragment shown in SEQ ID NO.7 at the XhoI / KpnI restriction site of the GV219 plasmid;

[0035] The internal control plasmid is an internal reference plasmid inserted into the ABL1 gene fragment shown in SEQ ID NO.8 at the XhoI / KpnI restriction site of the GV219 plasmid.

Embodiment 3

[0036] Example 3 The sensitivity detection of the positive control plasmid and the internal reference control plasmid of the present invention and the drawing of the fluorescence standard curve specifically include the following steps:

[0037] The first step is to carry out 10-fold gradient dilution of the positive control plasmid in Example 2 with sterilized double-distilled water for injection to obtain positive control plasmid standard solutions containing different SMIM3 gene fragment copy numbers. number is 10 6 、10 5 、10 4 、10 3 、10 2 ;

[0038] Carry out 10-fold gradient dilution of the internal reference control plasmid in Example 2 with sterilized double-distilled water for injection to obtain internal reference control plasmid standard solutions containing different ABL1 gene fragment copy numbers, and the ABL1 gene fragment copy numbers in each 1 μL internal reference control plasmid standard solution are sequentially for 10 6 、10 5 、10 4 、10 3 、10 2 ;

...

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Abstract

The invention discloses a primer probe set, kit and method for quantitative detection of an expression level of a small integral membrane protein 3 (SMIM3) gene. The primer probe set comprises a component A and a component B; the component A includes a forward primer SMIM3-FP, a reverse primer SMIM3-RP and a probe SMIM3-probe; and the component B includes a forward primer ABL1-FP, a reverse primerABL1-RP and a probe ABL1-probe. The primer probe set, the kit and the method have the advantages that quantitative detection of the relative expression level of the SMIM3 gene in a cell is realized;moreover, the sensitivity of the SMIM3 gene and the sensitivity of an ABL1 gene both reach up to 100 copies; the sensitivity is high, results are accurate, and the reliability is high; a new auxiliarydiagnosis method or auxiliary identification method is provided for hematologic tumor cells (especially acute myelogenous leukemia and acute lymphoblastic leukemia); and the primer probe set, the kitand the method have broader application prospects.

Description

technical field [0001] The present invention relates to the field of biochemical detection, in particular to a primer probe set for quantitative detection of SMIM3 gene expression, a kit containing the primer probe set, and a method for quantitative detection of SMIM3 gene expression . Background technique [0002] Small Integral Membrane Protein 3 is SMIM3, also known as C5orf62 or NID67. The protein encoded by the SMIM3 gene is a single-chain transmembrane protein. The mRNA of SMIM3 was highest in the heart, ovary and adrenal gland, while it was only expressed at a low level in most brain regions, testis, thyroid, thymus, pituitary, kidney and intestine, and there was almost no SMIM3 in skeletal muscle and cerebellum; but in PC12 In chromocytoma cells, SMIM3 can be preferentially induced by NGF and EGF, and SMIM3 can also be strongly induced by forskolin, A23187 and ATP. [0003] The human SMIM3 gene is located at 5q33.1, and the cDNA of SMIM3 contains an open reading fr...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6886C12Q1/6851C12Q2600/158C12Q2600/166C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 王树娟王冲刘延方姜中兴张雨李梦亚范熠李涛李亚飞
Owner THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV
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