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Application of poloxamer in improvement of reverse transcriptase efficiency or improvement of reverse transcriptase inhibitor tolerance and additive mixture

A reverse transcriptase inhibition and reverse transcriptase technology, applied in the biological field, can solve the problems of inhibitor residue and low RNA extraction, and achieve the effects of strong tolerance, improved sensitivity and accuracy, and high cDNA yield.

Pending Publication Date: 2021-08-10
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, RNA samples from COVID-19 are generally derived from nasopharyngeal swabs, anal swabs, or alveolar extracts, which are prone to problems such as low RNA extraction and serious inhibitor residues during the extraction process

Method used

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  • Application of poloxamer in improvement of reverse transcriptase efficiency or improvement of reverse transcriptase inhibitor tolerance and additive mixture
  • Application of poloxamer in improvement of reverse transcriptase efficiency or improvement of reverse transcriptase inhibitor tolerance and additive mixture
  • Application of poloxamer in improvement of reverse transcriptase efficiency or improvement of reverse transcriptase inhibitor tolerance and additive mixture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 tests RNA samples derived from normal cells.

specific Embodiment approach

[0042] The Total RNA used in the examples was extracted from cultured Human 293FT cells (GM-070006H, purchased from Jimon Biotechnology (Shanghai) Co., Ltd.) according to conventional methods, the same below. The specific implementation is as follows:

[0043] Table 2

[0044]

[0045]

[0046] Mix and spin away. 85°C for 5 minutes, and immediately placed on ice for 5 minutes.

[0047] table 3

[0048]

[0049] Mix and spin away. 10min at 25°C, 30min at 42°C, 5min at 85°C, hold at 4°C;

[0050] After diluting 1000 times, Hieff qPCR SYBR Green Master Mix (11200ES03) was used to quantify 18S rRNA and Actin RNA, and the quantitative primers are listed in Table 1. Quantitative results see figure 1 .

[0051] From the quantitative results, it can be seen that ( figure 1 ), different additives have a certain promotion effect on the efficiency of reverse transcription within a suitable range. Among them, the optimum concentration range of tetraethylammonium chlori...

Embodiment 2

[0052] Example 2 tests FFPE RNAA samples of different quality levels.

[0053] The FFPE RNA used in the examples comes from paraffin-embedded tissues, the same below. The specific implementation is as follows:

[0054] Table 4

[0055]

[0056]

[0057]Mix and spin away. 85°C for 5 minutes, and immediately placed on ice for 5 minutes.

[0058] table 5

[0059]

[0060] Mix and spin away. 10min at 25°C, 30min at 42°C, 5min at 85°C, hold at 4°C;

[0061] 18S rRNA and Actin RNA were quantified after 200-fold dilution, and the quantitative primers are listed in Table 1. Quantitative results see Figure 2-Figure 4 .

[0062] From the quantitative results, it can be seen that ( Figure 2-Figure 4 ), different additives have a significant effect on the reverse transcription efficiency of FFPE RNA in an appropriate range, especially for low-quality FFPE samples (DV200 is relatively small). Poloxamer 188 has the most obvious effect on promoting the reverse transcript...

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Abstract

The invention provides application of poloxamer in improvement of reverse transcriptase efficiency or improvement of reverse transcriptase inhibitor tolerance, and an additive mixture prepared by taking poloxamer as a core component and used in a reverse transcription process. The additive mixture not only can improve reverse transcription products (about 8 times) of normal high-purity RNA samples, but also has stronger tolerance and higher cDNA yield for RNA samples of complex sources, such as FFPE RNA or RNA containing reverse transcription inhibitors such as formaldehyde, ethanol, guanidine hydrochloride, guanidine isothiocyanate, heparin, tannic acid, formamide and phenol. The reverse transcriptase additive combination can significantly improve the sensitivity and accuracy of complex source pathological sample detection on RT-qPCR detection and RNA NGS library building, and is very suitable for the field of rapid diagnosis of diseases.

Description

technical field [0001] The patent of the present invention relates to the application of poloxamer in improving the efficiency of reverse transcriptase or improving the tolerance of reverse transcriptase inhibitors, and the additive mixture used in the process of reverse transcription, belonging to the field of biotechnology. Background technique [0002] RNA detection technology is an important means for pathogen detection and marker screening in the medical field. Reverse transcriptase-mediated RNA complementary cDNA synthesis is the first step in RNA detection technology. However, the reverse transcription efficiency of the current reverse transcriptase and system is low, and it is extremely sensitive to inhibitors in the reverse transcription system, such as formaldehyde and guanidine isothiocyanate. This seriously affects the sensitivity and accuracy of RNA detection technology and hinders the development of RNA detection technology. RNA from pathological sources, suc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6848
CPCC12Q1/6848C12Q2521/107C12Q2527/125
Inventor 江翱马海玲陈晶晶陆文佳曹振宋东亮
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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