Circulating RNAs were isolated from serum/plasma by S/P miRsol method developed in this study. Briefly, 1 ml of RNAiso-Plus (TaKaRa, Dalian, China) supplemented with 0.1 pM spiked-in Caenorhabditis elegans cel-miR-54-5p for normalization was added to 100 μl of serum or plasma and quickly vortexed thoroughly. Then 200 μl of chloroform was added and mixed by vigorous shaking for 20 s. The samples were incubated for 5 min at room temperature and centrifuged at 12,000 g for 15 min at 4 oC. A 500 μl of aqueous phase was transferred into a fresh tube and mixed with 6 μl of glycogen (AppliChem, Darmstadt, Germany). An equal volume (506 μl) of isopropyl alcohol was added into the mixture and incubated at −20 oC for 10 min followed by centrifugation at 13,500 g for 10 min at 4 oC. The RNA pellet was washed with 1 ml of 75% ethanol and dissolved in 20 μl of RNase-free water. The same procedure was used to isolate total RNAs from cultured cells except that no spiked-in cel-miR-54-5p and glycogen were used.
In certain experiments, second extraction was performed to see whether additional RNAs can be recovered from serum/plasma samples. Briefly, after 500 μl of aqueous phase was removed for the first exaction, an equal volume (500 μl) of RNase-free water was added to the organic phase. A 500 μl aqueous phase was collected as the second extraction of RNAs after mixing and centrifugation
In certain experiments, second extraction was performed to see whether additional RNAs can be recovered from serum/plasma samples. Briefly, after 500 μl of aqueous phase was removed for the first exaction, an equal volume (500 μl) of RNase-free water was added to the organic phase. A 500 μl aqueous phase was collected as the second extraction of RNAs after mixing and centrifugation
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