Method for extracting extracellular RNA from seminal plasma

[0030] Embodiment 1 Experimental data of the present invention

[0031] 1. Reagents used in experiments of the present invention

[0032] Diethyl pyrocarbonate Sigma (St.Louis, USA)

[0033] DNase I Fermentas Life Science (Hanover, USA)

[0034] MMLV Reverse Transcriptase Toyobo Company (Osaka, Japan)

[0035] RNase Inhibitor Toyobo Company (Osaka, Japan)

[0036] Olig(dT) 18 Toyobo Company (Osaka, Japan)

[0037] Taq TM DNA polymerase TaKaRa Biotechnology (Dalian, China)

[0038] Denaturing solution A 50mmol / L sodium citrate .2H 2 O (pH 7.0)

[0039] 8mol / L Guanidine Thiocyanate

[0040] 1% Sodium N-Lauryl Sarcosine (sarcosyl)

[0041] 2% (v / v) Triton X-100

[0042] 0.2mol / L β-mercaptoethanol (add just before use, add 14.4mol / L β-mercaptoethanol per ml

[0043] -Mercaptoethanol 14ul)

[0044] Denaturing solution B 300mmol / L sodium acetate (pH 5.

[0099] Methods: The seminal plasma of 12 cases was collected and separated, and the RNA was precipitated and washed with isopropanol and ethanol after two denaturing solutions. The OD260 value was measured with a nucleic acid analyzer, and the extracellular RNA concentration was calculated. Agilent2100 electrophoresis was used to observe the quality of the extracted extracellular RNA. The extracted extracellular RNA was used for reverse transcription-PCR to amplify the mRNA of housekeeping gene β-ACTIN, testis-specific expression gene hVASA, epididymis-specific expression gene hWFDC8, and prostate-specific expression gene hTGM4. Gel electrophoresis and sequencing identification to further test the quality of the extracted extracellular RNA.

[0100] Results: Extracellular RNA was successfully extracted from semen in all 12 cases, and the extracellular RNA content per milliliter of semen was 1.73±0.57ug. Agilent 2100 electrophoresis test results of the quality of the extracted R