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Rochel reverse-transcription reagent and preparation method thereof

A technology of reverse transcription and reverse transcriptase, applied in the field of cell biotechnology, can solve the problems of low hTERT efficiency, unsatisfactory effect, large hTERT gene fragments, etc., and achieves fast reverse transcription speed, easy promotion and use, and storage time. long effect

Inactive Publication Date: 2019-09-13
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] PDLCs cell lines can be constructed by overexpressing hTERT with lentivirus, but the hTERT gene fragment is large, the titer of recombinant virus is not high, and the expression hTERT efficiency is low, so the effect is not ideal

Method used

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  • Rochel reverse-transcription reagent and preparation method thereof
  • Rochel reverse-transcription reagent and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The invention provides a Rochel reverse transcription reagent, wherein the main materials used include by weight: deionized water 2ml, EASY Dilution for Real Time PCR1ml, gDNA Eraser0.1ml, 5x gDNA EraserBuffer0.2ml, RNase inhibitor 0.03 ml and 0.3ml of gene primers, said gene primers include telomerase reverse transcriptase 0.05ml, acid glyceraldehyde dehydrogenase 0.05ml, alkaline phosphatase 0.04ml, core binding factor 0.04ml, bone sialoprotein 0.03ml, Osteocalcin 0.03ml, osteopontin 0.02ml and type Ⅰ collagen 0.02ml;

[0034] Further, each primer sequence in the gene primer is specifically as follows:

[0035] Telomerase reverse transcriptase: F: 5'-TCTGGGATGCGAACGGGC-3'; R: 5'-TCCGGCTCAGGGGCAGC-3';

[0036] Glyceraldehyde phosphate dehydrogenase: F: 5’-GGAGTCCACTGGCGTCTTC-3’; R: 5’-GCTGATGATCTTGAGGCTGTTG-3’

[0037] Telomerase reverse transcriptase: F: 5'-CTCGTTGACACCTGGAAGAGCTTCAAACCG-3', R: 5'-GGTCCGTCACGTTGTTCCTGTTCAGC-3';

[0038] Core binding factor: F: 5'-CAC...

Embodiment 2

[0051] The invention provides a Rochel reverse transcription reagent, wherein the major materials used include by weight: 2.5ml of deionized water, 1.5ml of EASY Dilution for Real Time PCR, 0.2ml of gDNA Eraser, 0.3ml of 5x gDNA EraserBuffer, RNase Inhibitor 0.04ml and gene primer 0.4ml, described gene primer comprises telomerase reverse transcriptase 0.08ml, acid glyceraldehyde dehydrogenase 0.07ml, alkaline phosphatase 0.05ml, core binding factor 0.05ml, bone sialoprotein 0.04ml, osteocalcin 0.04ml, osteopontin 0.03ml and type Ⅰ collagen 0.03ml;

[0052] Further, each primer sequence in the gene primer is specifically as follows:

[0053] Telomerase reverse transcriptase: F: 5'-TCTGGGATGCGAACGGGC-3'; R: 5'-TCCGGCTCAGGGGCAGC-3';

[0054] Glyceraldehyde phosphate dehydrogenase: F: 5’-GGAGTCCACTGGCGTCTTC-3’; R: 5’-GCTGATGATCTTGAGGCTGTTG-3’

[0055] Telomerase reverse transcriptase: F: 5'-CTCGTTGACACCTGGAAGAGCTTCAAACCG-3', R: 5'-GGTCCGTCACGTTGTTCCTGTTCAGC-3';

[0056] Core bi...

Embodiment 3

[0069] The invention provides a Rochel reverse transcription reagent, wherein the main materials used include by weight: deionized water 3ml, EASY Dilution for Real Time PCR2ml, gDNA Eraser0.3ml, 5x gDNA EraserBuffer0.4ml, RNase inhibitor 0.05 ml and gene primer 0.5ml, said gene primer includes telomerase reverse transcriptase 0.1ml, acid glyceraldehyde dehydrogenase 0.1ml, alkaline phosphatase 0.06ml, core binding factor 0.06ml, bone sialoprotein 0.05ml, Osteocalcin 0.05ml, osteopontin 0.04ml and type Ⅰ collagen 0.04ml;

[0070] Further, each primer sequence in the gene primer is specifically as follows:

[0071] Telomerase reverse transcriptase: F: 5'-TCTGGGATGCGAACGGGC-3'; R: 5'-TCCGGCTCAGGGGCAGC-3';

[0072] Glyceraldehyde phosphate dehydrogenase: F: 5’-GGAGTCCACTGGCGTCTTC-3’; R: 5’-GCTGATGATCTTGAGGCTGTTG-3’

[0073] Telomerase reverse transcriptase: F: 5'-CTCGTTGACACCTGGAAGAGCTTCAAACCG-3', R: 5'-GGTCCGTCACGTTGTTCCTGTTCAGC-3';

[0074] Core binding factor: F: 5'-CACTATC...

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Abstract

The invention discloses a Rochel reverse-transcription reagent and a preparation method thereof, and specifically relates to the field of cell biotechnology. The Rochel reverse-transcription reagent comprises deionized water, EASY Dilution for Real Time PCR, gDNA Eraser, 5x gDNA Eraser Buffer, RNase inhibitor and gene primers; and the gene primers comprise telomerase reverse transcriptase, acid-glyceraldehyde dehydrogenase, alkaline phosphatase, core-binding factor, bone sialoprotein, osteocalcin, osteopontin and type-I collagen. Through arrangement of a plurality of combinatorial gene primers, the Rochel reverse-transcription reagent is capable of facilitating osteocyte reproduction; and fast reverse transcription is ensured during application. Moreover, high survival rate and good growthrate are achieved so as to facilitate study on periodontal membrane regeneration. In the process of production, aseptic treatment and low-temperature storage are adopted, so that, the the reverse-transcription reagent is good in quality and long in storage life; and thus, needs of DNA biosynthesis can be satisfied.

Description

technical field [0001] The invention relates to the field of cell biotechnology, more specifically, the invention relates to a Rochel reverse transcription reagent and a preparation method thereof. Background technique [0002] Periodontal ligament is a connective tissue that connects cementum and alveolar bone. There are various cell populations in periodontal ligament cells (PDLCs), mainly including fibroblasts, cementoblasts, osteoblasts, endothelial cells, and endothelial cells. cells, Malaseez epithelial cells, osteoclasts, etc. In recent years, studies have found that PDLCs have functions similar to mesenchymal stem cells (MSCs), and can differentiate into osteoblasts, adipocytes, and nerve-like cells in vitro, making periodontal tissue regeneration possible. Unfortunately, The mechanism of periodontal ligament regeneration is still unclear. The research on periodontal ligament regeneration is mainly limited by the subculture of primary PDLCs, which will reduce the d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/10
Inventor 张志东秦晓东何祥一曾飒
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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