Method for extracting small molecular RNA
An extraction method and small molecule technology, which is applied in the field of small RNA extraction, can solve the problems of toxicity and influence on microRNA analysis, and achieve good recovery effect
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Embodiment 1
[0023] Example 1 Extraction of microRNA in plasma:
[0024] Take 100 μl of fresh or -80°C frozen plasma and add it to a 1.5ml plastic centrifuge tube, add 100 μl of guanidine hydrochloride lysate to the centrifuge tube, vortex and mix for 10 seconds, then incubate at room temperature for 10 minutes, then add the mixture to a 30K ultrafiltration centrifuge tube (Pall Corporation, Cat. #OD030C33), centrifuged at 15000 rpm for 20 minutes at room temperature. Add 2.5 times of absolute ethanol to the centrifuge tube according to the volume after centrifugation, and mix well by blowing with a pipette. The mixed liquid was transferred to a nucleic acid purification column (Bioer 2 ml), and centrifuged at 13000 rpm for 1 minute. Discard the liquid in the collection tube, add 600 μl eluent (0.01M Tris-HCl pH 7.5, 0.2M NaCl, 0.2mM EDTA, 80% ethanol, the above reagents are all prepared with DEPC-treated purified water) into the column, centrifuge at 13000rpm for 1 minute. The liquid...
Embodiment 2
[0025] Example 2 MicroRNA extraction from tissues:
[0026] Take 50mg of adult liver tissue and grind it into powder in liquid nitrogen, add it to 2ml tissue lysate (4M guanidine isothiocyanate, 0.5% sodium lauryl creatine, 8% β-mercaptoethanol, 26mM sodium citrate), Vortex for 10 seconds, incubate at room temperature for 5 minutes, and centrifuge at 15,000 rpm for 10 minutes at room temperature. 500 μl of the supernatant was added to a 30K (Pall Corporation, Cat. #OD030C33) ultrafiltration centrifuge tube, and centrifuged at 15000 rpm for 20 minutes at room temperature. Add 2.5 times of absolute ethanol to the centrifuge tube according to the volume after centrifugation, and mix well by blowing with a pipette. The mixed liquid was transferred to a nucleic acid purification column and centrifuged at 13000 rpm for 1 minute. Discard the liquid in the collection tube, add 600 μl eluent (0.01MTris-HCl (pH 7.5), 0.2M NaCl, 0.2mM EDTA, 80% ethanol, all the above reagents are pre...
Embodiment 3
[0027] Example 3 MicroRNA detection:
[0028] In this example, the microRNAs obtained in Examples 1 and 2 were reverse-transcribed to synthesize miR-24 cDNA, and real-time quantitative PCR was performed to detect miR-24 therein. The specific method is as follows:
[0029]Take 12 μl of the microRNA extracted in Example 1 or 2 above and add it to a 1.5ml plastic centrifuge tube, add 1 μl of 5pm reverse transcription primer to the centrifuge tube, mix and place in a 70°C water bath for 5 minutes. Place quickly on ice for 5 minutes. Add 4 μl of 5× reverse transcription buffer (promega, 2800 Woods Hollow Road. Madison, WI 53711-399USA), 1 μl of 10 mM dN TPs, 1 μl of RNase inhibitor (40 activity units) and 1 μl of reverse transcriptase (200 activity units) into the centrifuge tube Unit) in a water bath at 42°C for 60 minutes, take out the centrifuge tube, and bathe in a water bath at 70°C for 10 minutes, and the obtained liquid is cDNA.
[0030] Take 1 μl cDNA, add 5×PCR buffer ...
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