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Internal reference for real-time quantitative PCR detection of serum and plasma miRNA

A real-time quantitative, plasma-based technology, applied in DNA/RNA fragmentation, recombinant DNA technology, microbial determination/inspection, etc., to achieve obvious social benefits, reduce psychological pressure, and enhance happiness.

Inactive Publication Date: 2015-11-11
HARBIN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Aiming at many problems such as insufficient reference, heterogeneity, insufficient stability and interspecies contamination in the process of relative quantification of miRNA at present, the present invention provides an internal reference miR-30e or miR-30e for real-time quantitative PCR detection of serum / plasma miRNA miR-30a overcomes the shortcomings and misunderstandings of the existing endogenous and exogenous internal reference standards, and provides a relatively simple and effective internal reference for the standardization of serum miRNA content

Method used

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  • Internal reference for real-time quantitative PCR detection of serum and plasma miRNA
  • Internal reference for real-time quantitative PCR detection of serum and plasma miRNA
  • Internal reference for real-time quantitative PCR detection of serum and plasma miRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 serum / plasma preparation

[0042] 1) Spare parts: vacuum sodium citrate or EDTA anticoagulant collection tube, or one vacuum blood collection tube without anticoagulant, one set of blood collection needle, tourniquet, iodine tincture, and 75% ethanol. 1 centrifuge, 1 set of pipettes, sterile pipette tips and several cryopreservation tubes.

[0043] 2) Before venous blood collection, check whether there are any defects in the blood collection equipment to ensure that the collection tube is not damaged, the anticoagulant is not deteriorated, there is no turbidity and foreign matter, and it is within the shelf life. Before blood collection, the blood collection tube should be shaken so that the anticoagulant can be fully coated on the tube wall.

[0044] 3) Collect venous blood: choose a thick, straight, and elastic vein, first disinfect it with iodine, then sterilize it with alcohol after 1 minute, then tie a tourniquet, insert the needle with the slope of t...

Embodiment 2

[0047] Example 2 Serum / Plasma RNA Extraction

[0048]1) Spare parts: enzyme-free 15mL and 1.5mL centrifuge tubes, absolute ethanol, chloroform, enzyme-free 1mL, 200uL, and 10uL pipette tips, 1 box each, 1 centrifuge tube rack, 1 set of pipettes, desktop low-temperature centrifugation 1 machine. One set of miRNeasySerum miRNA extraction kit produced by QIAGEN (Product No.: 217184). 1 bottle of RNaseZap, 1 pair of sterile masks and 1 pair of gloves.

[0049] 2) Take the serum / plasma sample out of the refrigerator, thaw it on ice, wear a mask and gloves during the period, and spray the operating table, pipette and related extraction consumables with RNaseZap to inactivate RNase.

[0050] 3) Use a pipette to transfer 200uL sample to a new 1.5mL enzyme-free centrifuge tube. Add 1 mL of QIAzolLysis Reagent. Vortex or pipette to mix. Incubate at room temperature (15–25°C) for 5 minutes.

[0051] 4) Add 200 uL of chloroform to the mixture, shake vigorously for 15 s, mix well and...

Embodiment 3

[0061] Example 3 RNA Quantitative Detection

[0062] 1) Spare parts: Agilent SmallRNAChip, enzyme-free 15mL and 1.5mL centrifuge tubes, enzyme-free deionized water, 1 bottle of RNaseZap, 1 box of enzyme-free 1mL, 200uL, 10uL pipette tips, 1 centrifuge tube rack, 1 set of pipettes , 1 desktop low-temperature centrifuge, 1 Agilent Bioanalyzer 2100 detector, 1 set of chip preparation station (Agilentprimingstation) with 1mL syringe.

[0063] 2) Instrument preparation: first draw 500uLRNaseZap and add it to a cleaning chip provided in the Agilent SmallRNAChip kit, pay attention to avoid bubbles when adding liquid. Place the cleaning chip on the chip installation platform of the detector, close the cover, soak the probe for 5 minutes, replace another chip pre-added with 500uL enzyme-free deionized water, and soak the cleaning probe until the detection starts.

[0064] 3) Before using the kit, about 650uL of liquid gel should be transferred to a centrifuge filter tube (provided in ...

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Abstract

The invention relates to an internal reference for real-time quantitative PCR detection of serum and plasma miRNA, and belongs to the technical field of biology. The internal reference for the real-time quantitative PCR detection of the serum and plasma miRNA is miR-30e or miR-30a. The invention discloses primers for detecting the internal reference. The sequences of a primer pair for amplifying miR-30e are shown in SEO ID NO:1 and SEQ ID NO:2, and the sequences of a primer pair for amplifying miR-30a are shown in SEO ID NO:3 and SEQ ID NO:2. The internal reference and the primers can be used for preparing a detection kit for the internal reference of serum and plasma miRNA and a labelled molecule kit for identifying prostate cancer prognosis miRNA. Expressions of the internal reference miR-30a and miR-30e are most stable among different individuals, the expressive abundance is high, and the internal reference is suitable for miRNA quantitative reference standard applied in serologic detection.

Description

technical field [0001] The invention relates to an internal reference for real-time quantitative PCR detection of serum / plasma miRNA, in particular to an internal reference miR-30e / a for real-time quantitative PCR detection of serum / plasma miRNA, belonging to the field of biotechnology. Background technique [0002] Single-stranded miRNA molecules with a length of 21-23bp can target and bind to the untranslated region of mRNA in a partially complementary manner, guide its degradation or inhibit the translation of mRNA, and cause post-transcriptional silencing of target genes. miRNA plays an important regulatory role in many physiological and pathological processes, and almost participates in the pathological process of all human malignant tumors. At present, it has been confirmed that miRNA in blood has potential application value in the diagnosis, treatment and prognosis evaluation of various malignant tumors, which has given birth to a new way of diagnosing and evaluating ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/118C12Q2600/166C12Q2600/178
Inventor 梁梅花王丽宏黄小义孙玉倩傅雪莲
Owner HARBIN MEDICAL UNIVERSITY
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