CN102817085B - Method for rapid reverse transcription of micro ribonucleic acid (RNA) library - Google Patents

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

The method in a kind of quick reverse transcription microRNA library

Technical field

The present invention relates to biology field, be specifically related to the method in a kind of quick reverse transcription microRNA library.

Background technology

The reverse transcription of miRNA is generally the synthetic Auele Specific Primer (as shown in Figure 1) of specific sequence design that uses loop-stem structure and contain 6 bases, but the specific binding due to its base, so method each time reverse transcription can only complete the reverse transcription of a kind or the identical miRNA of several terminal bases sequence, when detecting the expression of a plurality of miRNA, be, it is particularly numerous and diverse that this method just becomes, and need to design separately and reverse transcription each miRNA primer.

Summary of the invention

To the object of the invention is to the miscellaneous work amount increasing while needing a plurality of miRNA of reverse transcription in order overcoming, a kind of simple, method in reverse transcription microRNA library to be fast provided.

Above-mentioned purpose of the present invention is achieved by the following technical programs:

When reverse transcription, pass through to use Poly A tailing enzyme, end at all miRNA increases by one section of Poly A sequence, to in reverse transcriptase primer, change the Poly T sequence that can combine with Poly A sequence into the part of miRNA specific binding simultaneously, and will add Poly A tail and reverse transcription and be combined into a step and carry out, make the reverse transcription of all miRNA can use identical reverse transcriptase primer, thus the disposable reverse transcription that completes whole miRNA.

Compared with prior art, the present invention has following beneficial effect:

The present invention will add Poly A tail and reverse transcription and be combined into a step and carry out, and make the reverse transcription of all miRNA use identical reverse transcriptase primer, overcome the miscellaneous work amount increasing while needing a plurality of miRNA of reverse transcription, the experimental procedure of simplification.

Accompanying drawing explanation

Fig. 1 is traditional loop-stem structure; Wherein, 1.miRNA, 2. traditional reverse transcriptase primer, 3. reverse transcription gained cDNA;

Fig. 2 is Poly A tailing reverse transcription method schematic diagram; Wherein, 1.miRNA, 3. reverse transcription gained cDNA, 4. use Poly A tailing enzyme to add A tail, be 5. combined with reverse transcriptase primer and start reverse transcription, the reverse transcriptase primer that 6. the present invention uses, 7. use fluorescence quantitative PCR detection, 8.miRNA Auele Specific Primer, 9. reverse primer;

Fig. 3 is U6 amplification curve;

Fig. 4 is U6 solubility curve derivative figure;

Fig. 5 is miRNA150 amplification curve;

Fig. 6 is miRNA150 solubility curve;

Fig. 7 is miRNA200a amplification curve;

Fig. 8 is miRNA200a solubility curve;

Fig. 9 is miRNA30b amplification curve;

Figure 10 is miRNA30b solubility curve;

Figure 11 is RNA consumption and U6C (t) graph of a relation.

Figure 12 is RNA consumption and miRNA150 C (t) graph of a relation.

Figure 13 is RNA consumption and miRNA200a C (t) graph of a relation.

Figure 14 is RNA consumption and miRNA30b C (t) graph of a relation.

Embodiment

Below in conjunction with embodiment, further explain the present invention, but embodiment does not limit in any form to the present invention.

1. total RNA that the SW480 cell strain of take extracts is example, gets template ribonucleic acid and is placed on ice thaw (be whole RNA, need contain miRNA), and dNTP and 5 * buffer are placed in room temperature thaw (15-25 ℃).After it thaws completely, flick tube wall, liquid is mixed completely.Centrifugal liquid on tube wall is flowed down, then reagent is placed on ice and is deposited.

2. each formula of mixing as shown in the table, mixes to be placed on ice and preserves.

Composition	Volume/reaction
		5x buffer	4 ul
10mM dNTP	2 ul
		10mM ATP	1 ul
Rnase-free water	Variable
		Reverse Transcriptase	1 ul
Poly A Polymerase	1 ul
		Inhibitor	1 ul
Primer(is as shown in SEQ ID NO:5)	1ul
		U6 Primer(is as shown in SEQ ID NO:6)	1ul
Template RNA	Variable
		Total	20 ul

3. after will above each component mixing, put upside down gently and mix and centrifugal, be placed on ice and preserve.

4. hatch 60min for 37 ℃.

5. hatch 5min for 85 ℃, be placed on ice and preserve.

6. then can carry out real-time PCR as template, or be placed in-20 ℃ of freezing preservations (Fig. 2).

This reverse transcription method has been used real-time PCR to detect, uses respectively 200ng, 20ng, and 2ng, 0.2ng carries out reverse transcription, then uses fluorescence quantitative PCR detection, and U6 is that results of comparison is as shown in Fig. 3-14.U6(is as shown in SEQ ID NO:1) with miRNA150(as shown in SEQ ID NO:2) miRNA200a(is as shown in SEQ ID NO:3), miRNA30b(is as shown in SEQ ID NO:4) PCR result is unimodal, and as shown in Figure 11-14, U6 and miRNA150, miRNA200a, the slope of the Trendline of miRNA30b is all between-1.1 ~-0.9, so method can reflect the variation of concentration completely.

Applicant: Shenzhen Hong Kong University of Science and Thchnology of Peking University medical center

Denomination of invention: the method in a kind of quick reverse transcription microRNA library

 

SEQ?ID?NO:1

Title: U6

Sequence: gtgctcgctt cggcagcaca tatactaaaa ttggaacgat acagagaaga ttagcatggcccctgcgcaa ggatgacacg caaattcgtg aagcgttcca tatttt

 

SEQ?ID?NO:2

Title: miRNA150

Sequence: ucucccaacccuuguaccagug

 

SEQ?ID?NO:3

Title: miRNA200a

Sequence: uaacacugucugguaacgaugu

 

SEQ?ID?NO:4

Title: miRNA30b

Sequence: uguaaacauccuacacucagcu

 

SEQ?ID?NO:5

Title: primer

Sequence: GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACTTTTTTT-V-N

 

SEQ?ID?NO:6

Title: U6 primer

Sequence: CGCTTCACGAATTTGCGTGTCAT

Claims (1)

1. the method in quick reverse transcription microRNA library, is characterized in that, comprises the following steps:

S1. total RNA that the SW480 cell strain of take extracts is template, is placed on ice and thaws, and dNTPs and 5 * buffer are placed in room temperature and thaw, after it thaws completely, flick tube wall, liquid is mixed completely, centrifugal liquid on tube wall is flowed down, then reagent is placed on ice and is deposited;

S2. each formula of mixing as shown in the table:

Composition Volume/reaction 5x buffer 4μL 10mM dNTPs 2μL 10mM ATP 1μL Rnase-free water Variable ThermoScript II 1μL PolyA polysaccharase 1μL Inhibitor 1μL Primer shown in SEQ ID NO:5 1μL U6 primer shown in SEQ ID NO:6 1μL Template ribonucleic acid Variable Cumulative volume 20μL

S3. after will above each component mixing, put upside down gently and mix and centrifugal, be placed on ice and preserve;

S4. hatch 60min for 37 ℃;

S5. hatch 5min for 85 ℃, be placed on ice and preserve;

S6. then can carry out real-time PCR as template, or be placed in-20 ℃ of freezing preservations.