Technology for detecting tiny RNA155 (ribonucleic acid 155) relative content of T cells to reflect individual immunity state

A relative quantitative, mir-155 technology, applied in the fluorescent quantitative polymerase chain reaction, molecular biology field, can solve the problems of high throughput, long analysis time, high sample requirements, etc., to shorten the experimental time, reliable detection technology , the effect of reducing economic costs

Inactive Publication Date: 2011-08-31
陈必成 +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the deficiencies in the existing immunodiagnostic technology and molecular diagnostic technology in the evaluation technology of individual immune status, including defects such as long time-consuming analysis, incapable of high throughput, and high requirements for specimens

Method used

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  • Technology for detecting tiny RNA155 (ribonucleic acid 155) relative content of T cells to reflect individual immunity state
  • Technology for detecting tiny RNA155 (ribonucleic acid 155) relative content of T cells to reflect individual immunity state

Examples

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Embodiment 1

[0028] First, after wetting the injection syringe with heparin, take about 3ml of venous blood routinely, and turn the syringe to mix the blood evenly. Add whole blood to the culture bottle (5ml of 1640 culture solution containing 20% ​​serum, 5mg of PHA, pH 7.2) in the ultra-clean workbench, add 0.5ml of whole blood (13-15 drops of No. 7 needle), and tightly cover the rubber stopper , shake gently. Place the culture flask in a constant temperature incubator at 37°C for 16 hours. Collect uncultured and cultured blood cells in centrifuge tubes, and centrifuge at 1000rpm for 5min after balancing, remove the supernatant and leave 1ml, and mix thoroughly. Add 25 μL of T cell magnetic beads after mixing and invert repeatedly 2-3 times to disperse the magnetic beads. Gently rotate for 3 minutes at room temperature to allow the beads to attach to the T cells (do not exceed 4 minutes). Mixing can be done using an inversion device or by hand. Place on the magnetic stand for 3 minut...

Embodiment 2

[0030] A reverse transcription operation procedure for detecting miR-155 with this method. 2μL mRNA (0.5pg-1μg) in 20μL system, miR-155, U6 stem-loop primer mixture reagent A 2μL (final concentration 500nM), reverse transcription buffer reagent B 4μL (final concentration dNTP 1mM, M- MuLV reverse transcriptase concentration is 40U, Tris-HCl concentration is 50mM, KCl concentration is 50mM, MgCl 2 Concentration is 4mM), add water 12μL. The reverse transcription program was 5 minutes on ice, 15 minutes at 15°C, 15 minutes at 30°C, and 5 minutes at 95°C. After the reverse transcription is completed, 180 μL of water is added to the test tube to make a 10-fold dilution.

Embodiment 3

[0032] A Q-PCR method for detecting miR-155, as well as an operation process and a reaction program. For each specimen, 2 duplicate tubes for miR-155 detection and 2 duplicate tubes for U6 detection are required, a total of 4 reaction tubes, and the reaction volume of each reaction tube is 10 μL-50 μL. In 10 μL of Q-PCR system, 1 μL of diluted cDNA and 1 μL of specific primers (final concentration 200 nM) were firstly added. Add other reagents after mixing, including 4 μL of universal primer (final concentration 200 nM), 20 μL of Q-PCR buffer (final concentration 300 nM ROX, 0.5×SYBR GreenI dye, 1mM dNTPs, 40mM Tris-HCl, 40mM KCl, 20mM (NH 4 ) 2 SO 4 , 3mM MgSO 4 ), Taq enzyme 0.4 μL (final concentration 0.05U / μL), add water 8 μL. After mixing the reagents, add 8 μL of the mixed reagents to each well, cover the lid and perform detection on the ABI 7500. The procedure for cDNA amplification and quantification is as follows: 3 minutes of pre-denaturation at 95°C; 40-50 cy...

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Abstract

The invention discloses a stem-loop primer and a plurality of mating amplified primers, a kit and an assessment form, which can be used for carrying out quantitive detection on miR-155 (microRNA 155). The kit consists of a reagent A: 50mu mL of reverse transcription stem-loop primer mixed liquor; a reagent B: an M-MuLV (Moloney Murine Leukemia Virus) reverse transcriptase; a reagent C: a reverse transcription buffer solution; a reagent D: 100mu mL of universal primer; a reagent E: a miR-155specific primer; a reagent F: a U6 specific primer; a reagent G: Taq DNA (deoxyribose nucleic acid) polymerase; and a reagent H: a 2*PCR (Polymerase Chain Reaction) buffer solution. The kit provided by the invention can be used for carrying out relatively quantitive analysis on the miR-155 segments of different types of T cells, and assessing individual immunity states.

Description

technical field [0001] The invention relates to biotechnology, is a molecular biology method based on polymerase chain reaction (PCR), in particular relates to fluorescent quantitative polymerase chain reaction (Q-PCR). The relative content of microRNA-155 (microRNA-155, miR-155) reflects the individual's T cell immune status. The introduction of 8 specific nucleotide sequences at the 3' end of the stem-loop primer can reverse transcribe miR-155, and amplify miR-155 by Q-PCR technology. The relative content of miR-155 was derived after comparison with the content of U6 non-coding RNA. The relative content of miR-155 can reflect the immune status of individual T cells. This method can evaluate the immune status of individual T cells within 20 hours, and can analyze different T cell types through sorting technology to achieve a more accurate evaluation effect. After optimizing the experimental conditions, a simple and accurate relative quantitative detection kit for miR-155 w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 陈必成郑少玲杨亦荣王斯璐白永恒杨丽红
Owner 陈必成
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