Method for directly determining non-separated small nucleic acid in biological sample, and detection kit thereof

Abstract

The invention discloses a method for directly determining a non-separated small nucleic acid in a biological sample. The method comprises the following steps: 1, taking the biological sample; 2, adding Ploy A to the 3'-terminal of a small nucleic acid; 2, reverse transcription reaction: adding oligomerized dT with a first primer and retrovirus to generate single strand cDNA with oligomerized T+ and a designed first primer; and 4, PCR amplification of quantitative small nucleic acid: adding a second primer completely or partially complementary to the sequence of the small nucleic acid, and a third primer completely or partially complementary to the first primer, and carrying out PCR to detect the amount of the quantitative small nucleic acid. The content of the small nucleic acid (like siRNA, miRNA, antisense nucleic acid and aptamer) in the biological sample is determined by directly using the biological sample without separating the small nucleic acid in the biological sample.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method and a kit for quantitatively analyzing small nucleic acids in biological samples. Background technique [0002] Small nucleic acids are widely used in biomedical research and drug development, but the problem that needs to be solved urgently is: the tissue quantitative detection of siRNA is the bottleneck of the distribution and metabolic kinetics of small nucleic acids in vivo, and the distribution and metabolism of small nucleic acids in tissues are directly Or indirectly determine the evidence of the effect and toxicity of its small nucleic acid and delivery system, so it is urgent to establish a simple and accurate siRNA quantitative detection method. However, there is no good method for the determination of the amount of small nucleic acids in biological samples, especially the quantitative analysis of in vivo samples. The quantification of sm...

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Problems solved by technology

[0002] Small nucleic acids are widely used in biomedical research and drug development, but the problem that needs to be solved urgently is: the tissue quantitative detection of siRNA is the bottleneck of the distribution and metabolic kinetics of small nucleic acids in vivo, and the distribution and metabolism of small nucleic acids in tissues are directly Or indirectly determine the evidence of the effect and toxicity of its small nucleic acid and delivery system, so it is urgent to establish a simple and accurate siRNA quantitative detection method

However, the sensitivity of the above method is difficult to achieve the quantitative ability of small nucleic acid in plasma and tissue pharmacokinetic terminal elimination phase (concentration is usually lower than 10nmol / L).

In addition, the above two mainstream analysis methods also have a common defect, that is, the sample pretreatment process is quite complicated, the absolute recovery rate is low, the analysis period is long, the cost is high, and the application is limited.

The detection sensitivity of Enzyme-linked bridging assay (ELBA) for nucleic acid molecular hybridization is only slightly higher than the above methods, and it still cannot meet the precise requirements for the quantification of small nucleic acids in biological samples. Stem-loop RT-qPCR technology has been It is widely used, but according to the verification of our and other laboratories, the consistency of the experimental data is poor, the error is large, and it is difficult to meet the requirements of RNAi drug declaration

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