Method for extracting micro-RNA from plasma and kit thereof
A technology of plasma and reagents, which is applied in the field of methods and kits to extract microRNA, can solve the problems of high price and low extraction volume, and achieve the effect of improving recovery efficiency
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Embodiment 1
[0042] A method for extracting microRNA in human plasma, according to the following steps:
[0043] (1) After collecting human anticoagulated whole blood and mixing it, centrifuge at 500rpm for 10 minutes to remove blood cells, and obtain plasma samples and store them at -80°C for later use.
[0044] (2) Take 200 μL of the above plasma sample in a 1.5 mL centrifuge tube, add 200 μL of 10% sodium dodecyl sulfate (SDS) for 1:1 mixing, then add 1 μL of proteinase K 20 mg / ml, and heat at 75°C for 5 minutes; ℃ warm bath digestion 1h.
[0045] (3) Add 2 times the volume of Trizol reagent to the treated sample, mix well, and let stand at room temperature for 5 minutes; add 20% volume of chloroform, shake for 15 seconds, let stand for 2 minutes; centrifuge at 12,000 rpm and 4°C for 15 minutes, and take the supernatant .
[0046] (4) Add an equal volume of isopropanol and 1 μL of 15 mg / ml glycogen, mix well, and let stand overnight at -80°C.
[0047] (5) Centrifuge at 12000rpm at 4°...
Embodiment 2
[0050] A method for extracting microRNA in human plasma, according to the following steps:
[0051] (1) After collecting human anticoagulated whole blood and mixing it, centrifuge at 500rpm for 10 minutes to remove blood cells, and obtain plasma samples and store them at -80°C for later use.
[0052] (2) Take 200 μL of the above plasma sample into a 1.5 mL centrifuge tube, add 200 μL of 10% sodium dodecyl sulfate (SDS), and add 1 μL of proteinase K 20 mg / ml at the same time, shake and mix, heat at 75 °C for 5 minutes, and then heat at 42 °C Incubate for 1 hour.
[0053] (3) Add 2 times the volume of Trizol reagent to the treated sample, mix well, and let stand at room temperature for 5 minutes; add 20% volume of chloroform, shake for 15 seconds, let stand for 2 minutes; centrifuge at 12,000 rpm and 4°C for 15 minutes, and take the supernatant .
[0054] (4) Add an equal volume of isopropanol and 1 μL of 15 mg / ml glycogen, mix well, and let stand at -80°C overnight (8-12 hour...
Embodiment 3
[0058] A method for extracting microRNA in human plasma, according to the following steps:
[0059] (1) Collect 200 μL of human plasma, add 1 μL of proteinase K 20 mg / ml and an equal volume of 10% SDS, shake and mix.
[0060] (2) Heating at 75°C for 5 minutes, then incubating at 42°C for 1 hour.
[0061] (3) Add 2 times the volume of Trisol reagent and 1 / 5 times the volume of chloroform, shake and mix, and let stand for 15 minutes.
[0062] (4) Centrifuge at 12,000 rpm for 15 minutes at 4°C, take the supernatant, add an equal volume of isopropanol and 1 μL of 15 mg / ml glycogen.
[0063] (5) Stand overnight at -80°C, centrifuge at 12,000 rpm for 10 minutes at 4°C.
[0064] (6) The precipitate was collected, washed with 75% ethanol, and centrifuged at 12000 rpm for 5 minutes at 4°C.
[0065] (7) Collect the precipitate, dry it at room temperature, add 14 μL RNase-free water to dissolve it, and store it at -80°C.
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