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Small RNA detection method and use thereof

A detection method and small nucleic acid technology, applied in the field of small nucleic acid detection, can solve the problems of narrow detection range, high detection limit and high cost, and achieve the effects of low detection cost, high detection efficiency and strong specificity

Active Publication Date: 2014-08-27
BIOMICS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Previous studies have reported a variety of quantitative methods, including Elsia analysis (Kim EJ et al., J Control Release, 2010, 143(1):80-7.), radiolabeled hybridization analysis (Jin J et al., Biotechniques. 2010 Jun; 48(6): xvii-xxiii.), etc., these methods are time-consuming, expensive, narrow detection range, high detection limit and other shortcomings
Various PCR methods based on miRNA (a short single-stranded RNA), such as end-tailing, phosphate linker ligation, Stem-Loop PCR method (Chen C et al., Nucleic Acids Res. 2005;33(20):e179 .), due to the structural differences between synthetic siRNA and endogenous siRNA, neither can solve the precise quantification of siRNA well

Method used

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  • Small RNA detection method and use thereof
  • Small RNA detection method and use thereof
  • Small RNA detection method and use thereof

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Experimental program
Comparison scheme
Effect test

Embodiment l

[0042] Identifying the Role of Blocking Nucleic Acids in Small Nucleic Acid Detection

[0043] 1 Main reagents, materials and instruments

[0044] 1.1 The si-NC sense strand and antisense strand (see Table 1 for the sequence) were synthesized by Biomaike Biotechnology Co., Ltd. Dilute the sense strand (single-strand RNA, ssRNA) and the annealed double strand (double-strand RNA, dsRNA) of si-NC to a concentration of 1 μM, respectively.

[0045] Table 1. si-NC sequences

[0046] 1.2

[0047] 1.3 The closed nucleic acid (DNA sequence) of si-NC: according to the 5'-3' direction, the closed nucleic acid complementary to the antisense strand of si-NC is designed, the length is 7nt, the sequence is shown in Table 2, provided by Biomaike Biotechnology Co., Ltd. synthesis.

[0048] Table 2. Blocking nucleic acids for si-NC

[0049] Blocked Nucleic Acid Name Sequence (5'-3') Length (nt) NC-B1 AATTCTC (SEQ ID NO: 3) 7 NC-B2 CGAACGT (SEQ ID NO: 4) 7 ...

Embodiment 2

[0071] Detection of double-stranded small nucleic acid (taking si-NC as an example)

[0072] 1 Main reagents, materials and instruments

[0073] 1.1 Sense and antisense strand sequences of si-NC (Table 1), synthesized by Biomaike Biotechnology Co., Ltd.

[0074] 1.2 The closed nucleic acid (DNA sequence) of si-NC: according to the 5'-3' direction, the closed nucleic acid complementary to the antisense strand of si-NC is designed. synthesis.

[0075] 1.3 Quantitative PCR detection primers were synthesized by Biomaike Biotechnology Co., Ltd.

[0076] Forward primer: 5'-TGCCCTTCTCCGAACGTGTC-3' (SEQ ID NO: 6);

[0077] Reverse primer: 5'-GTGCAGGGTCCGAGGT-3' (SEQ ID NO: 7);

[0078] Reverse transcription stem-loop primer: 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAACGTGA-3' (SEQ ID NO: 8).

[0079] 1.4 Reagents: EzOmics TM One-Step qPCR kit (Biomicro Biotechnology Co., Ltd.), etc.

[0080] 1.5 Instruments: real-time quantitative PCR instrument (Bio-Rad Company); centr...

Embodiment 3

[0097] Detection of small nucleic acids with different ends

[0098] 1 Main reagents, materials and instruments

[0099] 1.1 The 3' end of the si-NC sense strand is UU's si-NC-M1 (Table 5), which was synthesized by Biomaike Biotechnology Co., Ltd. The sense and antisense strands were annealed into double strands and diluted to a concentration of 10 μM.

[0100]Table 5. Modified si-NC sequences

[0101] 1.2

[0102] 1.3 The closed nucleic acid (DNA sequence) of si-NC: according to the 5'-3' direction, the closed nucleic acid complementary to the antisense strand of si-NC is designed, the length is 7nt, the sequence is shown in Table 2, provided by Biomaike Biotechnology Co., Ltd. synthesis.

[0103] 1.4 Quantitative PCR detection primers were synthesized by Biomaike Biotechnology Co., Ltd.

[0104] Forward primer: 5'-TGCCCTTCTCCGAACGTGTC-3' (SEQ ID NO: 6);

[0105] Reverse primer: 5'-GTGCAGGGTCCGAGGT-3' (SEQ ID NO: 7);

[0106] Reverse transcription stem-loop primer: 5...

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Abstract

The present invention relates to a nucleic acid detection method and its application, especially the detection of double-stranded small nucleic acids, characterized in that the method is specifically designed to design several continuous or discontinuous closed nucleic acids complementary to one strand of the small nucleic acid , a reverse transcription stem-loop primer complementary to the 3' end of the other strand of the small nucleic acid, and a forward primer and a reverse primer for quantitative PCR, through denaturation, blocking, reverse transcription and quantitative PCR methods to detect small nucleic acid. The invention has the advantages of simple operation, low detection cost, detection limit up to femtogram level, high detection efficiency, strong specificity, high sensitivity and the like. The present invention can be applied to the quantitative analysis, qualitative analysis, pharmacokinetic analysis of small nucleic acid drugs and the amplification, specific identification and identification of small nucleic acid molecules, especially in molecular diagnosis, small nucleic acid drug metabolism and small nucleic acid drug function research. Wide range of uses.

Description

technical field [0001] The invention relates to a method for detecting small nucleic acids and applications thereof, in particular to a method for detecting double-stranded small nucleic acids and applications thereof. Background technique [0002] RNA interference (RNA interference, RNAi) is an ancient biological phenomenon that has been discovered in recent years and is commonly found in organisms. It is mediated by double-stranded RNA (double-stranded RNA, dsRNA) and participated by specific enzymes. The phenomenon of specific gene silencing, which blocks the expression of genes at the transcriptional, post-transcriptional, and translational levels. [0003] With the development of RNAi research, RNAi has become the basis of gene function research, drug target discovery and drug development. RNAi drugs are expected to become a more efficient and faster way to treat diseases. Small interfering RNA (small interfering RNA, siRNA) ) as the effector molecule of RNAi, it has m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 陆毅祥朱远源吉怡吴美华宋翅宇李铁军
Owner BIOMICS BIOTECH
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