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Extraction method for tiny RNA in serum or plasma

An extraction method and plasma technology, applied in the field of microRNA extraction, can solve the problems of limited large-scale application and high price, and achieve the effect of being beneficial to large-scale clinical verification and subsequent clinical application, and the extraction cost is low.

Active Publication Date: 2010-12-15
NINGBO AJCORE BIOSCIENCES INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First of all, the extraction of miRNAs in serum or plasma is a key step. Some existing miRNAs extraction kits can be used for the extraction of miRNAs in serum or plasma, such as the miRNeasy mini kit from Qiagen in the United States, but its price is relatively expensive and its size is limited. scale application

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  • Extraction method for tiny RNA in serum or plasma
  • Extraction method for tiny RNA in serum or plasma
  • Extraction method for tiny RNA in serum or plasma

Examples

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Comparison scheme
Effect test

Embodiment 1

[0030] The extraction of microRNA in embodiment 1 serum

[0031] A method for extracting microRNA in serum, the specific steps of which are as follows:

[0032]Thaw the serum sample pre-stored in the -70°C refrigerator on ice. After thawing, take 200 μl of the serum sample into a marked 1.5ml centrifuge tube ①, then add 400 μl of trizol reagent, vortex and mix and let stand for 8 ~10 minutes; then add 100μl chloroform to No. ① centrifuge tube, vortex and mix, put it into the centrifuge, centrifuge at 12000g / min, 4°C for 15 minutes; take out No. ① centrifuge tube from the centrifuge, absorb the upper white liquid Add 100μl chloroform to the ③ centrifuge tube of 1.5ml, shake and mix, put it into the centrifuge, centrifuge at 12000g / min, 4℃ for 15 minutes; take the ③ centrifuge tube out of the centrifuge , absorb the white liquid in the upper layer, which is the pre-separated sample;

[0033] Collect the pre-separated samples in 1.5ml centrifuge tube ②, and add 50 μl of magneti...

Embodiment 2

[0036] Embodiment 2 The present invention compares the effect of extracting microRNA in serum with other methods

Embodiment 3

[0066] The repeatability experiment of the extraction of microRNA in embodiment 3 serum

[0067] Thaw the serum sample pre-stored in -70°C refrigerator on ice, take 200 μl into a labeled 1.5ml centrifuge tube after thawing, and use the same method as in Example 1 to extract microRNA. Repeat three times, and the extracted samples containing microRNA are recorded as sample 1, sample 2, and sample 3 respectively. Sample 1, sample 2 and sample 3 extracted from three repeated experiments were analyzed by fluorescent quantitative PCR, and the results are shown in Table 1.

[0068] Wherein, the detection conditions and steps of fluorescent quantitative PCR are the same as in Example 2, and the detection is repeated three times, and the detection results are shown in Table 2. Fluorescence threshold (threshold) adopts 30000.

[0069] Table 2

[0070]

[0071] It can be seen from Table 2 that the extraction of microRNA in serum by the method of the present invention has very good ...

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Abstract

The invention discloses an extraction method for effectively enriching tiny RNA in serum or plasma, which comprises the following steps: preseparating the tiny RNA in a sample with Trizol and chloroform, capturing the preseparated tiny RNA in the sample by a method of magnetic beads marked with -Si-OH functional groups, separating the magnetic beads from the sample by a magnetic separator, washing and drying simply, adding RNA-free enzyme water, heating and eluting by a water bath, and further separating by the magnetic separator. The method of the invention can be used for the large-scale verification of the tiny RNA which is a candidate disease maker and the extracting of the tiny RNA during clinical practices, can efficiently enrich the tiny RNA in serum or plasma, is convenient and fast and has low price.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for extracting microRNA in serum or plasma. Background technique [0002] MicroRNAs (microRNAs, miRNAs) are a class of non-coding regulatory single-stranded small RNAs with a total length of 19-25 ribonucleotides, which are produced by shearing a single-stranded RNA precursor with a hairpin loop structure. The complementary pairing of the 3-terminal non-coding region (3-UTR) of the target mRNA (messenger RNA, messenger RNA) molecule inhibits the translation of the target mRNA molecule, thereby regulating the expression level of the target gene after transcription. [0003] According to the records of the miRBase database (the microRNA database), more than 9,500 miRNAs have been discovered. Studies have confirmed that many miRNAs are related to the occurrence and development of diseases, and it is particularly worthy of attention that they are closely related to t...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 郑智国毛伟敏牟瀚舟凌志强
Owner NINGBO AJCORE BIOSCIENCES INC
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