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An RT-qPCR method for the direct quantitative detection of circulating miRNAs

A quantitative detection and detection method technology, applied in the field of biomedicine, can solve the problems of incomplete RNA precipitation and recovery, time-consuming RNA extraction process, RNA loss, etc., to achieve excellent transcription efficiency, improve sensitivity and accuracy, and easy operation Effect

Active Publication Date: 2020-12-22
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Existing miRNA detection methods are based on purified RNA as a template. Due to the incomplete precipitation and recovery of RNA in the extracted nucleic acid, some RNA will inevitably be lost.
In addition, the RNA extraction process is time-consuming, prone to contamination and degradation
[0006] It can be seen that the existing technology still needs to be perfected

Method used

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  • An RT-qPCR method for the direct quantitative detection of circulating miRNAs
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  • An RT-qPCR method for the direct quantitative detection of circulating miRNAs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1 Direct S-Poly(T)Plus method compares the circulating miRNA content in plasma and serum

[0054] In this example, the same volunteer serum and plasma were used as templates at the same time, and a total of 10 pairs of serum and plasma samples from the same healthy volunteer were collected. Use the DirectS-Poly(T)Plus method in the present invention to detect the miRNA expression levels in the same amount of serum or plasma samples respectively. Specifically include the following steps:

[0055] S1, cracking and centrifuging, the specific steps are:

[0056] 1) Mix 20uL plasma / serum with 20uL 2×lysis buffer, add 1uL proteinase K, treat at 50°C for 20 minutes, then keep at 95°C for 5 minutes, and place on ice;

[0057] 2) Centrifuge at 13,000g at 4°C for 5 minutes; absorb the supernatant (crude RNA) and transfer it to another new centrifuge tube or use it directly in S2;

[0058] S2. Reverse transcription with tailing: poly(A) tailing and reverse transcript...

Embodiment 2

[0088] Embodiment 2 Comparison of the effects of different cracking schemes in the Direct S-Poly (T) Plus (DSPP) method of the present invention

[0089] In the Direct S-Poly(T)Plus method, any of the following six treatments can be used to cleavage the miRNA from the protein complex:

[0090] ① Lysis system: 20ul lysate, 20ul sample; lysis condition: keep at 75°C for 5 minutes;

[0091] ② Lysis system: 20ul RNase-free water, 1ul proteinase K, 20ul sample; lysis conditions: treat at 50°C for 20 minutes, then keep at 95°C for 5 minutes;

[0092] ③ Lysis system: 20ul lysate, 1ul proteinase K, 20ul sample; lysis conditions: treat at 50°C for 20 minutes, then keep at 95°C for 5 minutes;

[0093] ④ Lysis system: 20ul 2×lysis buffer, 20ul sample; lysis condition: keep at 75°C for 5 minutes;

[0094] ⑤ Lysis system: 20ul 2×lysis buffer, 1ul proteinase K, 20ul sample; lysis conditions: treat at 50°C for 20 minutes, then keep at 95°C for 5 minutes;

[0095] ⑥ Lysis system: 10ul 2×ly...

Embodiment 3

[0099] One-step method and two-step method sensitivity contrast in embodiment 3 Direct S-Poly (T) Plus method

[0100] In the previously invented S-Poly(T)Plus method (patent application number: 201510558101.5), using purified RNA as a template, the sensitivity of the one-step method is greatly improved compared with the two-step method. The two-step method means that miRNA Poly(A) tailing is completed before reverse transcription; the one-step method means that miRNA Poly(A) tailing and reverse transcription are performed in the same reaction. In this invention, using crudely extracted RNA as a template, the operation is the same as in Example 1, and the sensitivity of the two-step method and the one-step method are compared again. Such as image 3 Shown, in Direct S-Poly (T) Plus method, the scheme of the present invention makes the sensitivity of one-step method improve 2.5~52 times (1.7~5.7 Ct value difference) than its two-step method ( image 3 ).

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Abstract

The invention relates to the field of biomedicine, in particular to a real-time fluorescent quantitative RT-qPCR method for directly detecting circulating microRNA (miRNA) in serum or plasma without extracting nucleic acid. The method includes: S1: lysing exosomes and miRNA protein complexes in serum or plasma, and centrifuging to obtain crude circulating miRNA extracts; S2: miRNA tailing and reverse transcription; S3: RT-qPCR quantitative detection. In the miRNA direct fluorescent quantitative RT-qPCR amplification method [Direct S-Poly(T) Plus, DSPP for short] of the present invention, nucleic acid does not need to be extracted, and the Poly(A) tailing and reverse transcription of miRNA will be in one reaction system Synchronously completed, easy to operate and shorten the time, the preparation of cDNA can be completed within 95 minutes. Compared with the stem-loop method, the sensitivity of this technical system is increased by tens or even hundreds of times, and a very simple, sensitive, efficient, fast and cheap miRNA detection technical system has been established. This technical system is especially suitable for clinical application promotion and detection of miRNA from biological fluid samples with low miRNA abundance.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a RT-qPCR method for directly quantitatively detecting circulating miRNA without extracting nucleic acid. Background technique [0002] MicroRNA (miRNA) is a kind of non-coding small RNA with a length of about 22 nucleotides, which widely exists in eukaryotic organisms such as animals, plants, and nematodes. miRNAs regulate gene expression at the post-transcriptional level by binding to the 3'-untranslated region (3'-UTR) of target mRNAs, degrading target mRNAs or preventing their translation. Functionally, miRNAs are widely involved in cell differentiation, proliferation, apoptosis, individual growth and development, and organ formation. The expression of miRNAs in organisms is finely regulated with strict spatiotemporal specificity. Studies have found that circulating miRNA exists in the blood and is very stable. More importantly, the abnormality of circulating miRNA is closely rel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/686
CPCC12Q1/686C12Q2521/107C12Q2521/537C12Q2561/101C12Q2525/107C12Q1/6876
Inventor 苟德明牛燕琴康康
Owner SHENZHEN UNIV
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