Process for extracting free mRNA from pleural fluid and ascitic fluid

A technique for ascites and pleural effusion, which is applied in biochemical equipment and methods, microbial determination/examination, sugar derivatives, etc., can solve the problems of inaccurate diagnosis, difficulty in definite diagnosis, and low detection rate, and is conducive to early diagnosis. , The effect of strong repeatability and easy operation

Inactive Publication Date: 2007-04-25
THE AFFILIATED DRUM TOWER HOSPITAL MEDICAL SCHOOL OF NANJING UNIV
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Problems solved by technology

However, due to the relatively low pathological detection rate of tumor cells and the need for a certain number of pleural effusion and ascites samples, patients with pleural and peritoneal effusions often need to be repeatedly sent for examination in clinical practice, so it is difficult to make a clear diagnosis in time or because there are not enough samples. Due to the large number of sp

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  • Process for extracting free mRNA from pleural fluid and ascitic fluid
  • Process for extracting free mRNA from pleural fluid and ascitic fluid

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Embodiment Construction

[0025] 1. All experimental items are treated with RNase removal: related reagents are prepared with inactivated 0.1% DEPC water; the glassware used in the experiment needs to be baked at 160°C for 6 hours;

[0026] 2. Inject 5ml of pleural fluid or / ascites fluid into the RNase-free tube with 0.5ml of 3.8% sodium citrate added in advance;

[0027] 3. Centrifuge at 4000g for 10 minutes at 4°C, and transfer the supernatant to a new tube;

[0028] 4. Centrifuge again at 4000g for 10 minutes at 4°C, absorb 2 / 3 of the supernatant, and transfer to a new tube;

[0029] 5. Take 1ml of the supernatant, add it to 3ml Trizol LS (invitrogen product), shake at room temperature for 15 minutes, until the protein particles are fully lysed;

[0030] 6. Add chloroform, shake vigorously and place at room temperature for 15 minutes;

[0031] 7. Put on a 12000g centrifuge, centrifuge at 4°C for 15 minutes, and transfer the aqueous layer to another new tube;

[0032] 8. Add isopropanol to the new...

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Abstract

The invention concerns a method for extracting free mRNA from pleural fluid and abdominal dropsy. In the invention injecting pleural fluid and abdominal dropsy into an anticoagulation tube with natrium citricum without RNase, centrifugating, imbibing the supernatant, centrifugating again, imbibing the supernatant, adding the supernatant into Trizol LS solution, shaking at room temperature, adding chloroform, standing at room temperature, centrifugating and imbiling water phase layer to a new tube, adding isopropyl alcohol for RNA precipitation, centrifugating, washing with 75% ethanol, air-drying, adding RNase-free water, and dissolving RNA anti-transcribing with the RNA as templates using conventional technology into cDNA for preservation. The invention dissolves the problem that RNA can not be extracted form pleural fluid and abdominal dropsy in the past. The invention can provide early treatment for cancer and other diseases. The invention has the advantages of easy operation and high reproducibility.

Description

technical field [0001] The invention relates to a biotechnology extraction method, in particular to a method for extracting free mRNA from chest and ascites. Background technique [0002] Before the present invention, because pleural effusion and ascites were often malignant tumor metastasis or the first clinical manifestation, it was often done in medical practice to extract a certain volume of pleural and ascites for malignant tumor detection. However, due to the relatively low pathological detection rate of tumor cells and the need for a certain number of pleural effusion and ascites samples, patients with pleural and peritoneal effusions often need to be repeatedly sent for examination in clinical practice, so it is difficult to make a clear diagnosis in time or because there are not enough samples. Due to the large number of specimens, it is impossible to accurately diagnose and delay the best treatment opportunity; at the same time, in the detection of tumor-associated...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C07H21/02
Inventor 刘宝瑞王立峰钱晓萍
Owner THE AFFILIATED DRUM TOWER HOSPITAL MEDICAL SCHOOL OF NANJING UNIV
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