Cell one-step real-time quantitative PCR method, related reagent kit and applications

A real-time quantitative and kit technology, applied in the field of PCR detection, can solve the problems of small safety hazards and low operation difficulty, and achieve the effect of improving efficiency and strong consistency

Pending Publication Date: 2019-03-29
NOVOPROTEIN SCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the traditional RNA extraction method, this lysis method has the advantages of saving time and labor (only 10-20 minutes), low operational difficulty, small safety hazards, and low cost.

Method used

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  • Cell one-step real-time quantitative PCR method, related reagent kit and applications
  • Cell one-step real-time quantitative PCR method, related reagent kit and applications
  • Cell one-step real-time quantitative PCR method, related reagent kit and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 The direct cell lysing method of the present invention and the comparison of traditional RNA extraction method

[0042] 1.1 Cell direct lysis qPCR detection

[0043]Prepare 1× lysis buffer with DEPC water: 5mM Tris-Hcl (pH 8.0) + 1-20mM EDTA + 0.1%-2% Triton X-100 + 0.2-2U RNase Inhibitor (available from Wujiang Jinan Protein Technology Co., Ltd.) +0.1M DTT. Resuscitate CHO-DG44 cells and subculture for 3 times. After cell counting, the cells were transferred to EP tubes, and the supernatant was discarded after centrifugation. Add 1× Lysis Buffer (the final cell density is 300 cells / ul); after mixing, place at room temperature for 5-10 minutes, and centrifuge to remove cell debris. The Primer 5.0 software was used to design the intron-spanning primers for the reference gene gapdh in CHO-DG44 cells, and the specificity was detected by Blast. Take 1ul supernatant to 8 tubes, add 2ul 1st Strand cDNASynthesis SuperMix (available from Wujiang Nearshore Pro...

Embodiment 2

[0052] Embodiment 2 direct lysing method cell number detection range and the establishment of standard curve

[0053] Cells with a density of 2000 cells / ul were taken and diluted logarithmically with 1×PBS, and the dilution densities were as follows: 1000, 500, 250, 125, 62.5, 31.25, 15.625 cells / ul. Add 10× lysis buffer to lyse at room temperature for 5 minutes, and the supernatant after high-speed centrifugation is used for one-step RT-qCPR detection. The RT-qPCR program is detailed in Table 2. See Table 1 for primer sequences.

[0054] Such as figure 2 The standard curve of the direct cell lysis method shown, where the abscissa is log 10 (number of cells), the ordinate is the Ct value.

[0055] The results of this experiment show that 15-2000 cell lysates can be used for RT-qPCR detection and log 10 (Number of cells) has a linear relationship with the Ct value. It proves that the direct lysis method can detect the expression level of the target gene in a wide range of...

Embodiment 3

[0056] Example 3 Detection of the activating effect of blinatumomab on Jurkat cells by direct lysis method

[0057] 3.1 Cell plating and antibody activation

[0058] Jurkat cells were counted, and the cells were centrifuged and resuspended at 0.3×10 6 96-well plate at density per ml; Raji cells were evenly pipetted and counted at 0.3×10 5 cells / ml density into wells pre-coated with Jurkat. Plate overnight, add different concentrations of blinatumomab (purchased from Amgen) antibodies, and incubate overnight.

[0059] 3.2 Detection of activation of blinatumomab antibody by direct lysis method

[0060] After the cells were evenly pipetted, the suspension was pipetted into an EP tube, and 1 / 10 volume of 10× lysis buffer was added. Lyse at room temperature for 5 minutes, and take the supernatant after high-speed centrifugation for one-step RT-qPCR detection. Primer 5.0 software was used to design il-2 and the reference gene gapdh, and Blast was used for specific detection. T...

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Abstract

The invention provides a cell one-step real-time quantitative PCR method, a related reagent, applications and a reagent kit. The cell one-step real-time quantitative PCR method provided by the invention comprises the following steps: step a, extracting target cells, adding a lysis buffer, performing uniform mixing, performing standing at room temperature, and performing centrifuging to remove cellfragments to obtain a supernatant; step b, carrying out trans-intron design on reference genes of the target cells, and detecting specificity; and step c, adding a reaction system liquid into the supernatant of the step a, and carrying out one-step RT-qPCR detection. The reverse transcription and qPCR reagents and procedures are combined into the one-step RT-qPCR. On the one hand, the method develops a lytic cell, and the lytic supernatant can be directly used in the lysis buffer of the RT-qPCR. Compared with traditional RNA extraction method, the lysis method has the advantages of time and labor saving, low operation difficulty, small potential safety hazard and low cost. The method can be applied to various experimental tests such as comparison of monoclonal expression differences and biological activity effect of proteins or antibodies on cells.

Description

technical field [0001] The invention belongs to the field of molecular and cell biology, in particular to PCR detection. Background technique [0002] RT-PCR (Real time-polymerase chain reaction, real-time PCR) is a method based on PCR technology that can detect gene expression levels in real time, and is now widely used in drug function identification, signaling pathway research and in vitro diagnosis It is the most important tool for detecting gene transcription levels in cells. With the widespread application of biotechnology in the diagnostic and pharmaceutical industries, the detection of limited gene expression differences in multiple patient cells, the detection of the effects of multiple candidate drugs on the same target on the expression of certain genes, and the production of macromolecular drugs Stable cell lines with high expression of high transcriptional active sites have higher requirements for relatively large-scale detection of gene transcription levels. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851
CPCC12Q1/6851
Inventor 葛泰根江华超石加加任加庆臧赢
Owner NOVOPROTEIN SCI INC
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