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Application of microRNA in preparation of kit for diagnosis of cervical carcinoma or precancerous lesions

A technology of precancerous lesions and diagnostic reagents, applied in the field of biomedicine, can solve the problems of few applied researches and low sensitivity

Active Publication Date: 2016-04-13
HANGZHOU DALTON BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the 1990s, liquid-based cytology (liquid-based cytology, LBC) examination began to be developed. LBC realized the thin-layer preparation of liquid-based cells through special equipment, which improved the satisfaction rate of cervical exfoliated cell specimens. The sensitivity of CIN is still low, only 38-65%, which is not significantly different from the conventional Pap smear
Unfortunately, there are still not many studies on the application of miRNA (especially miRNA in cervical exfoliated cells) detection in the screening of cervical cancer or its precancerous lesions
In addition, new miRNAs related to cervical cancer or its precancerous lesions need to be further explored

Method used

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  • Application of microRNA in preparation of kit for diagnosis of cervical carcinoma or precancerous lesions
  • Application of microRNA in preparation of kit for diagnosis of cervical carcinoma or precancerous lesions
  • Application of microRNA in preparation of kit for diagnosis of cervical carcinoma or precancerous lesions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Example 1, Screening of cervical cancer-related miRNA markers

[0106] 1. Microarray primary screening of miRNA expression differences

[0107] The present invention first uses the miRNA chip to compare and analyze the miRNA expression profiles in cervical exfoliated cells (n=6) with normal histology, high-grade CIN (CIN2-CIN3), and squamous cell carcinoma (SCC) ( figure 1 ). Using ANOVA analysis, from 875 human genome miRNAs (SangermiRBase release13.0), 31 miRNAs with significant expression differences were screened as potential markers for the diagnosis of cervical cancer or its precancerous lesions. Among them, 14 miRNAs (let-7b, miR-145, miR-126, miR-199a, miR-195, miR-29a, miR-375, miR-10b, miR-29c, miR-218, miR-424 , miR-100, miR-125b, miR-99a) were down-regulated in cervical cancer or its precancerous lesions, and 17 miRNAs (miR-155, miR-92a, miR-92b, miR-224, miR-221 , miR-222, miR-31, miR-182, miR-106a, miR-17, miR-20a, miR-20b, miR-15b, miR-16, miR-25, miR-...

Embodiment 2

[0143] Embodiment 2, the preparation of miRNA detection kit

[0144] 1. Kit based on RT-qPCR technology

[0145] In this embodiment, the miRNA detection kit based on nucleic acid amplification includes the following components:

[0146] (1) total RNA extraction reagent: Trizol solution, chloroform, isopropanol, ethanol, DEPC water;

[0147] (2) miRNA reverse transcription reagents: reverse transcriptase, RNase inhibitor, reverse transcription buffer, dNTP mixture.

[0148] (3) miRNA reverse transcription primers: miR-375 reverse transcription primer and miR-424 reverse transcription primer, reverse transcription primer of U6 used as internal reference.

[0149] (4) PCR detection reagents for miRNA: Taq enzyme, Mg 2+ , PCR buffer, dNTP mix, nucleic acid dye.

[0150] (5) miRNA nucleic acid amplification primers: miR-375qPCR upstream primers, miR-424qPCR upstream primers, miRNAqPCR general downstream primers. U6 upstream and downstream primers used as internal reference.

...

Embodiment 3

[0166] Example 3, miRNA detection based on hybrid capture method

[0167] The detection kit for miR-375 or miR-424 was performed using the hybrid capture-based miRNA detection kit prepared in Example 2 above.

[0168] (1) Sample treatment: take clinical samples and mix them with sample lysate (same as in Example 2), and incubate in a 46° C. water bath for 45-60 minutes.

[0169] (2) Nucleic acid hybridization: take out the lysed sample from the water bath and cool to room temperature. Take a 96-well microplate as a hybridization microplate, add the lysed sample and single-stranded DNA probe reagent, and seal the hybridization microplate with a sealing film. Then place the hybridization microplate on a microplate heating shaker, shake at 1100rpm for 3-5 minutes, and mix well. Incubate at 46°C for 60 minutes, then remove the hybridization microplate and cool to room temperature. The presence of miR-375 or miR-424 in the sample will combine with the single-stranded DNA probe t...

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Abstract

The invention relates to an application of microRNA in preparation of a kit for diagnosis of cervical carcinoma or precancerous lesions. According to the invention, obvious low expression of miR-375 and miR-424 in tissues and cells of cervical carcinoma or precancerous lesions is revealed, and the low expression is obviously relative to the cervical carcinoma or precancerous lesions. The invention also provides a cervical carcinoma or precancerous lesions diagnosis method and the kit for determining existence amount of miR-375 and / or miR-424.

Description

technical field [0001] The invention belongs to the field of biomedicine; more specifically, it relates to the application of microRNA in the preparation of a kit for diagnosing cervical cancer or its precancerous lesions, providing assistance for the clinical shunting of HPV-positive women and the detection of cervical cancer or its precancerous lesions diagnosis. Background technique [0002] Cervical cancer is one of the most common female malignancies worldwide. According to the International Agency for Research on Cancer (IARC) of the World Health Organization, there were about 529,000 new cases of cervical cancer in the world in 2008, 85% of which occurred in underdeveloped regions, and 59% occurred in Asia; the death cases reached 275,000, of which 85% of deaths occurred in developing countries. According to data from 72 tumor registries of the China Cancer Center, the incidence rate of cervical cancer registered in China in 2009 was 12.96 / 100,000, and the incidence...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574
Inventor 韩斌谢幸吕卫国程晓东王新宇胡杰锋华绍炳
Owner HANGZHOU DALTON BIOSCI
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