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Human small RNS 129 fluorescent quantitative reagent box

A technology of fluorescence quantification and reagent kits, which is applied in the direction of measuring devices, microbial measurement/inspection, and material analysis through optical means, which can solve difficult problems and achieve the effect of simple design and production

Inactive Publication Date: 2008-02-20
HANGZHOU FIRST PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the length of hsa-miR-129 is only 21nt, it is difficult to design common PCR to detect hsa-miR-129

Method used

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  • Human small RNS 129 fluorescent quantitative reagent box
  • Human small RNS 129 fluorescent quantitative reagent box
  • Human small RNS 129 fluorescent quantitative reagent box

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment one kit standard curve preparation

[0042] A positive control and a negative control should be set up for each test. Dilute the standard with sterile deionized water to 1×10 2 -1×10 7 copy / ul.

[0043] 1. Synthesis of standard cDNA 10ul artificially synthesized hsa-miR-129 template (1×10 2 copies / μl-1×10 7 copy / μl (six gradients) add 50ng / ul stem-loop reverse transcription primer 129RT-primer1.0ul, denature at 70°C for 10 minutes, and immediately put in ice bath; then add 10mMdNTP 1.0ul, 5×RT buffer4.0ul, RNase inhibitor 1.0ul, dithiothreitol 1.0ul, SuperScript II reverse transcriptase 1.0ul, add dH2O to the reaction system to 20ul; room temperature for 10 minutes, 42°C for 45 minutes, 85°C for 5 minutes, 4°C for 5 minutes .

[0044] 2. Fluorescent polymerase chain reaction Take 5.0ul of the above reverse transcription product for PCR amplification according to the following system: 5.0ul 10×PCR buffer, 8.0ul 25mM Mg 2+ , 1.0ul 10mMdNTP, upstream pri...

Embodiment 2

[0048] Example 2 Detection of hsa-miR-129 expression in bladder cancer and normal tissues

[0049] A positive control and a negative control should be set up for each test. Dilute the standard with sterile deionized water to 1×10 2 -1×10 7 copy / ul.

[0050] 1. Extraction of total RNA Total RNA from bladder cancer tissue and corresponding normal bladder mucosa tissue was extracted using TrizolReagent (Invitrogen, Carlsbad, CA) extraction was carried out as follows:

[0051] (1) Tissue plus Trizol  After the reagent was ground on ice, it was left at room temperature for 5 min to fully lyse.

[0052] (2) Centrifuge at 12000rpm for 5min, and discard the precipitate.

[0053] (3) Add chloroform to 200ul chloroform / ml Trizol, shake and mix for 15 minutes, and place at room temperature for 15 minutes.

[0054] (4) Centrifuge at 12000 g for 15 min at 4°C.

[0055] (5) Absorb the upper aqueous phase into another centrifuge tube.

[0056] (6) Add isopropanol to 0.5ml isopr...

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PUM

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Abstract

The utility model relates to an artificial RNA129 detection reagent box, comprising a reverse transcriptase and reaction buffer solution, a stem ring reverse transcription primer, a Taq DNA polymerase, a PCR mixed solution, a PCR primer with fluorescent quantification and probe, an artificially synthesized hsa-miR-129 standardized goods and reference substance, wherein the reverse transcription reaction solution comprises a reverse transcription primer 129RT-primer, a reverse transcription buffer solution and a dNTPs. The polymerase chain reaction solution comprises a polymerase chain reaction buffer solution, an MgCl2, a dNTPs, an upstream primer for detection, a down stream primer for detection and a fluorescent probe. The utility model can detect the hsa-miR-129 simply, practically with quantification, the sensitivity of which is 1x102Copy / ul and can become the clinical auxiliary quantitative detection measures for diagnosing the tumor or other diseases and one of the important methods for researching the disease mechanism in future.

Description

technical field [0001] The invention belongs to the field of biological technology, and is a cDNA obtained by reverse transcription (RT) microRNA samples, combined with real-time fluorescent quantitative PCR detection technology, can accurately and quantitatively detect hsa-microRNA-129 (hsa-miR-129, human small RNA 129) expression kit. Background technique [0002] microRNA (miRNA) is a newly discovered small non-coding RNA molecule with a length of about 22 nt. It comes from the intergenic region or broken intron of genomic DNA. It does not encode protein itself. Currently, 587 species have been found in humans. miRNA (microrna.sanger.av.uk / targets / v4), hsa-miR-129 is one family. The discovery of miRNA was rated as one of the top ten technological breakthroughs in 2002 by the American "Science" magazine, and was ranked first. [0003] The miRNA gene is first transcribed to produce a primary product (pri-miRNA) of hundreds to thousands of nt, which is cut into a hairpin p...

Claims

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Application Information

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IPC IPC(8): G01N21/00C12Q1/68
Inventor 王刚张红河
Owner HANGZHOU FIRST PEOPLES HOSPITAL
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