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Kit for detecting changes of lung cancer cell RNA

A kit and lung cancer technology, applied in the direction of DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of high-sensitivity molecular markers, lack of specificity, etc.

Inactive Publication Date: 2013-01-30
杨祚璋
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The lack of specific and highly sensitive molecular markers is currently the key problem restricting the early diagnosis and effective treatment of lung cancer bone metastases.

Method used

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  • Kit for detecting changes of lung cancer cell RNA
  • Kit for detecting changes of lung cancer cell RNA
  • Kit for detecting changes of lung cancer cell RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Changes in tumor cell microRNA expression before and after bone metastasis of lung cancer

[0023] 1. Materials and methods

[0024] 1. Materials

[0025] The tumor cells were all from the hospitalized cases of lung cancer in Yunnan Cancer Hospital from 2004 to 2010. The tumor cells of 30 patients with bone metastasis of lung cancer and the tumor cells of 30 patients with non-metastasis of lung cancer were collected respectively.

[0026] 2. Method

[0027] 2.1 Extraction of total RNA from tumor cells before and after bone metastasis of lung cancer

[0028] According to the instructions of the Trizol kit, RNA was extracted from tumor cells of lung cancer patients with bone metastases and non-metastatic lung cancer patients. The integrity of RNA was verified by gel electrophoresis, and the concentration and purity of RNA were determined with a nucleic acid protein analyzer. The total RNA extraction kit was used by Shanghai Huashun Bioengineering Co., Ltd. to...

Embodiment 2

[0074] Example 2 Expression analysis of microRNA30 in patients with bone metastasis of lung cancer and normal people

[0075] 1. Experimental materials

[0076] The blood of 50 lung cancer patients with bone metastases and 50 normal people were collected respectively to verify whether novel_mir_30 was expressed in lung cancer patients with bone metastases and the changes in the expression level.

[0077] 2. Experimental methods and results

[0078] 1 Primer design and synthesis

[0079] According to the sequence information of novel_mir_30, four primers were designed. The first one is the reverse transcription primer RT30 with a stem-loop structure, whose sequence is shown in the sequence listing SEQ NO.1; the second is the upstream specific primer F30, whose sequence is shown in the sequence listing SEQ NO.2; the third is the downstream universal primer R30, whose sequence is shown in the sequence listing SEQNO.3; the fourth is the fluorescent probe primer Flu30 needed in f...

Embodiment 3

[0095] Example 3 Preparation and use of a fluorescent quantitative PCR detection kit for lung cancer bone metastases

[0096] 1. Composition of novel_mir_30qRT-PCR kit

[0097]

[0098] 2. Detection of novel_mir_30qRT-PCR

[0099] 2.1 Preparation of serum small RNA

[0100] The blood of 120 patients with bone metastases from lung cancer and 100 normal people were selected. Centrifuge at 12,000 rpm for 10 minutes, and collect the supernatant.

[0101] 2.2 cDNA synthesis

[0102] The obtained serum was subjected to reverse transcription reaction, and the reverse transcription system was: reverse transcription primer RT30 SEQ ID NO.1 (2 μmol / L) 1 μl, blood supernatant 4 μl, dNTP mixture (each 2.5mmol / L) 4 μl, 0.1mol / LDTT 2 μl, SuperScript RNase H reverse transcriptase (200 U / μl) 2 μl (purchased from Invitrogen Company) The reaction conditions are: 37° C. water bath for 60 minutes, 95° C. for 3 minutes.

[0103] 2.3qRT-PCR detection

[0104] qRT-PCR was amplified on a Na...

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Abstract

The invention discloses a kit for detecting changes of lung cancer cell RNA. The kit comprises the following components: specific primers, a specific probe, a standard DNA template and a fluorescent quantitative PCR reaction solution. The invention also discloses a usage method of a fluorescence quantitative PCR kit for detection of bone metastasis of lung cancer. The kit can rapidly conduct quantitative detection on expression of novel_mir_89, so as to detect bone metastasis of lung cancer. The invention has the advantages of simple operation, rapidness, stable result, high sensitivity and strong specificity.

Description

technical field [0001] The present invention relates to the preparation and use method of small RNA detection kits, specifically, the present invention relates to the design of specific oligonucleotide primers and fluorescent labeling probes based on the newly discovered nucleotide sequence of small RNA precursors , using real-time fluorescent quantitative PCR technology to assemble a detection kit for bone metastasis of lung cancer, and the present invention also relates to a detection method thereof. Background technique [0002] Lung cancer is the leading cause of cancer death in the world today, and its morbidity and mortality are on the rise, seriously threatening human health and life. In 2000, there were 1.2 million new lung cancer cases and 1.1 million deaths worldwide. The main reason for the high mortality rate of lung cancer is that it is difficult to detect early lung cancer due to asymptomatic symptoms. Lung cancer is prone to bone metastases, and studies have...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 杨祚璋谢琳徐磊李国奇
Owner 杨祚璋