Baseline zero dnase treatment

For characterizing the effect of freeze thaw on cell free mRNA expressions, RNA was extracted using plasma processed with S1, S2, S1FR, S2FR, and S1FRS2 conditions. Cell free mRNA was isolated by using plasma/serum circulating and exosomal RNA purification Kit (Norgen Biotek, cat. 42800) followed by 10X Baseline-ZERO DNase treatment (Epicentre, cat. DB0715K). DNase treated RNA samples were purified and further concentrated using RNA clean and concentrator (Zymo Research, cat. R1014). The purified RNA samples were assayed by RT-qPCR using custom selected 16 primers targeting MTND2, PPBP, B2M, PF4, ACTB, CORO1C, GSE1, GAPDH, SMC4, HBG1, NUSAP1, MIKI67, FGB, APOE, FGG, and ALB. Template RNA was mixed with Superscript III One-step RT-PCR system with Platinum Taq DNA polymerase (Invitrogen, cat. 11-732-020) to generate cDNA according to the protocol. PCR amplification products were treated with Exonuclease I (New England Biolabs, cat. M0293L) to digest single stranded primers at 37 °C for 30 min followed by inactivation of enzymes at 80 °C for 15 min. For RT-qPCR, cDNA from preamplification was diluted 1:80 and set-up in 96-well plates with SsoFast EvaGreen supermix with low ROX (BioRad, cat. 1725211) with above primers at 10 µM. QuantStudio 7 Flex (Applied Biosystems) was used to run RT-qPCR assay according to manufacturer’s recommended cycling conditions.