Subject area	Biochemistry, Genetics and Molecular Biology
More specific subject area	MicroRNA isolation
Protocol name	One-phase phenol-free method for microRNA isolation from blood plasma
Reagents/tools	-lysis buffer (5 M guanidine isothiocyanate, 0.75 M NaCl, 0.5 % SDS, 5 % Triton X100, 20 mM TrisHCl, pH = 8) -1 M DTT -1 μg/μl poly(A)RNA (or 1 μg/μl yeast tRNA) -5 μg/μl LPAA -isopropanol -1 wash buffer (60 % isopropanol, 10 mM TrisHCl, pH = 8.0) -2 wash buffer (75 % ethanol, 10 mM TrisHC,l pH = 8.0) -RNase-free water
Experimental design	To select the best reagents composition 6 comparative isolations from 8 plasma samples were performed. The following variables were tested: the ratio of the plasma sample volume to the lysis buffer volume, the molarity of NaCl in the lysis buffer, the effect of replacement of β-mercaptoethanol by dithiothreitol and effect of the addition of LPAA and RNA (poly(A)RNA and tRNA) as coprecipitator. As a result, we got the protocol that showed better recovery efficiency: 4 volumes of lysis buffer containing 0.75 M NaCl and adding 2.5 μM DTT, 10 μg poly(A)RNA, 50 μg of LPAA per sample. In comparison with miRNeasy Mini Kit, our protocol showed better yield (Δ median Ct 4.94 for cel-238, р = 1,0E-04 and Δ median Ct 2.18 for microRNA-21, р = 9,0E-04).
Trial registration	–
Ethics	All donors were familiar with the content of the work and signed informed consent.