Small RNA detection method and use thereof

A detection method and technology for small nucleic acids, which are applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of high detection limit, narrow detection range, time-consuming, etc., and achieve high detection efficiency, low detection cost, Sensitive effect

Active Publication Date: 2013-06-05
BIOMICS BIOTECH
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  • 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
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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 si-NC closed nucleic acid (DNA sequence): according to the 5'-3' direction, the closed nucleic acid complementary to the si-NC antisense strand is designed, the length is 7nt, the sequence is shown in Table 2, provided by Biomaike Biotechnology Co., Ltd. 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. ...

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 invention relates to a small RNA detection method and a use thereof and belongs to the field of double-stranded small RNA detection. The small RNA detection method comprises the following steps of 1, designing multiple continuous or discontinuous closed RNAs which are complementary with one chain of a small RNA, a reverse transcription stem-loop primer which is complementary with an end 3' of the other chain of the small RNA, and a forward primer and a reverse primer for quantitative PCR, and 2, detecting the small RNA by denaturation, enclosing, inverse transcription and quantitative PCR. The small RNA detection method has simple processes, a low detection cost, a detection limit reaching a femtogram grade, high detection efficiency, strong singularity and high sensitivity. The small RNA detection method can be widely used for quantitative analysis, qualitative analysis and pharmacokinetic analysis of a small RNA drug, for amplification, specific identification and differentiation of a small RNA molecule, and especially for molecular diagnosis, small RNA drug metabolism and small RNA drug functional study.

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 Applications(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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