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Method for rapid reverse transcription of micro ribonucleic acid (RNA) library

A reverse transcription and library technology, applied in the field of molecular biology, can solve complicated problems, achieve the effect of simplifying experimental steps and overcoming complicated workload

Inactive Publication Date: 2014-07-16
SHENZHEN PKU HKUST MEDICAL CENT
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Problems solved by technology

[0002] The reverse transcription of miRNA generally uses a stem-loop structure and a specific sequence containing 6 bases to design and synthesize specific primers (such as figure 1 shown), but due to the specific combination of bases, this method can only complete the reverse transcription of one or several miRNAs with the same terminal base sequence each time. When it is necessary to detect the expression of multiple miRNAs, This approach becomes particularly cumbersome, requiring individual design and reverse transcription of each miRNA primer

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  • Method for rapid reverse transcription of micro ribonucleic acid (RNA) library
  • Method for rapid reverse transcription of micro ribonucleic acid (RNA) library
  • Method for rapid reverse transcription of micro ribonucleic acid (RNA) library

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Embodiment Construction

[0022] The present invention is further explained below in conjunction with the examples, but the examples do not limit the present invention in any form.

[0023] 1. Taking the total RNA extracted from SW480 cell line as an example, take template RNA and thaw it on ice (it is all RNA and must contain miRNA), and thaw dNTP and 5×buffer at room temperature (15-25°C). Once it is completely thawed, flick the tube wall to mix the liquid completely. Centrifuge to drain the liquid from the walls of the tube, then store the reagents on ice.

[0024] 2. Mix the formulations as indicated in the table below and store on ice after mixing.

[0025] Element volume / reaction 5x buffer 4 ul 10mM dNTPs 2 ul 10mM ATP 1 ul RNase-free water Variable Reverse Transcriptase 1 ul Poly A Polymerase 1 ul Inhibitor 1 ul Primer (as shown in SEQ ID NO:5) 1ul U6 Primer (as shown in SEQ ID NO:6) 1ul Template RNA Variable Total ...

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Abstract

The invention discloses a method for rapid reverse transcription of a micro ribonucleic acid (RNA) library. The method includes: polymeric aluminum (PolyA) tailing enzyme is used during the reverse transcription, one section of PolyA sequence is respectively added at the tail ends of all miRNAs, simultaneously a portion combined with miRNA specificities in a reverse transcription primer is replaced by a PolyT sequence combined with the PolyA sequences, PolyA tailing and reverse transcription are performed in one step, all reverse transcription can use the same reverse transcription primer, and therefore reverse transcription of all miRNAs can be completed at one time. The method for the rapid reverse transcription of the micro ribonucleic acid (RNA) library can simplify experimental procedures greatly, brings convenience for experiments, and is suitable for popularization and application at laboratories.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for rapid reverse transcription of a microRNA library. Background technique [0002] The reverse transcription of miRNA generally uses a stem-loop structure and a specific sequence containing 6 bases to design and synthesize specific primers (such as figure 1 shown), but due to the specific combination of bases, this method can only complete the reverse transcription of one or several miRNAs with the same terminal base sequence each time. When it is necessary to detect the expression of multiple miRNAs, This approach becomes particularly cumbersome, requiring individual design and reverse transcription of each miRNA primer. Contents of the invention [0003] The purpose of the present invention is to provide a simple and fast method for reverse transcribing a microRNA library in order to overcome the increased complicated workload when reverse transcribing multiple mi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C12N15/113C12N15/10
Inventor 万峻杨旸张超于波关明张伟董小林
Owner SHENZHEN PKU HKUST MEDICAL CENT
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