TY - JOUR
T1 - A TRIzol-based method for high recovery of plasma sncRNAs approximately 30 to 60 nucleotides
AU - Rodgers, Kristen P.
AU - Hulbert, Alicia
AU - Khan, Hamza
AU - Shishikura, Maria
AU - Ishiyama, Shun
AU - Brock, Malcolm V.
AU - Mei, Yuping
N1 - Publisher Copyright:
© 2022, The Author(s).
PY - 2022/12
Y1 - 2022/12
N2 - Protein functional effector sncRNAs (pfeRNAs) are approximately 30–60 nucleotides (nt), of which the extraction method from plasma has not yet been reported. Silver staining in a high-resolution polyacrylamide gel suggested that the majority of plasma sncRNAs extracted by some broadly used commercial kits were sncRNAs from 100 nt upwards. Additionally, TRIzol’s protocol is for long RNA but not sncRNA recovery. Here, we report a TRIzol-based frozen precipitation method (TFP method), which shows rigor and reproducibility in high yield and quality for plasma sncRNAs approximately 30–60 nt. In contrast to the yields by the commercial kit, plasma sncRNAs extracted by the TFP method enriched more sncRNAs. We used four different pfeRNAs of 34 nt, 45 nt, 53 nt, and 58 nt to represent typical sizes of sncRNAs from 30 to 60 nt and compared their levels in the recovered sncRNAs by the TFP method and by the commercial kit. The TFP method showed lower cycle threshold (CT) values by 2.01–9.17 cycles in 38 plasma samples from 38 patients, including Caucasian, Asian, African American, Latin, Mexican, and those who were a mix of more than one race. In addition, pfeRNAs extracted by two organic-based extraction methods and four commercial kits were undetermined in 22 of 38 samples. Thus, the quick and unbiased TFP method enriches plasma sncRNA ranging from 30 to 60 nt.
AB - Protein functional effector sncRNAs (pfeRNAs) are approximately 30–60 nucleotides (nt), of which the extraction method from plasma has not yet been reported. Silver staining in a high-resolution polyacrylamide gel suggested that the majority of plasma sncRNAs extracted by some broadly used commercial kits were sncRNAs from 100 nt upwards. Additionally, TRIzol’s protocol is for long RNA but not sncRNA recovery. Here, we report a TRIzol-based frozen precipitation method (TFP method), which shows rigor and reproducibility in high yield and quality for plasma sncRNAs approximately 30–60 nt. In contrast to the yields by the commercial kit, plasma sncRNAs extracted by the TFP method enriched more sncRNAs. We used four different pfeRNAs of 34 nt, 45 nt, 53 nt, and 58 nt to represent typical sizes of sncRNAs from 30 to 60 nt and compared their levels in the recovered sncRNAs by the TFP method and by the commercial kit. The TFP method showed lower cycle threshold (CT) values by 2.01–9.17 cycles in 38 plasma samples from 38 patients, including Caucasian, Asian, African American, Latin, Mexican, and those who were a mix of more than one race. In addition, pfeRNAs extracted by two organic-based extraction methods and four commercial kits were undetermined in 22 of 38 samples. Thus, the quick and unbiased TFP method enriches plasma sncRNA ranging from 30 to 60 nt.
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U2 - 10.1038/s41598-022-10800-0
DO - 10.1038/s41598-022-10800-0
M3 - Article
C2 - 35474236
AN - SCOPUS:85128985468
SN - 2045-2322
VL - 12
JO - Scientific reports
JF - Scientific reports
IS - 1
M1 - 6778
ER -