Sds absolute quantification software version 2

Total small RNA was extracted from serum with mirVana™ PARIS™ Kit according to the instructions from the manufacturer (Ambion, AM1556, Austin, TX). The eluate containing the microRNA was stored at −20°C, until use. Five microliters of the eluate was used for the reverse transcription reaction. For generation of standard curves, chemically synthesized miR-122 was dissolved in distilled H₂O and diluted to varying concentrations (10²–10⁶ copies/µl). Input RNA was reverse transcribed using the TaqMan miRNA Reverse Transcription Kit and miRNA-specific stem-loop primers (Applied BioSystems, Carlsbad, CA) in the reverse transcription reaction. Measurement of serum miR-122 was performed using real-time PCR on an Applied BioSystems 7900HT thermocycler and data computed with SDS Absolute Quantification Software Version 2.2.2 (Applied BioSystems).

Total human genomic DNA (hgDNA) was extracted from serum by QIAamp DNA Blood Mini kit. The single copy gene beta-actin was chosen to represent hgDNA and was measured with a Taqman real-time PCR assay. Known 10 ng/µl concentrations of hgDNA were purchased from Applied BioSystems and used at varying dilutions in the real-time PCR reaction (3030, 303, 30.3, 3, 1.0 copies/µl) to generate a standard curve for estimating serum hgDNA levels. Measurement of serum hgDNA was performed using real-time PCR on an Applied BioSystems 7900HT thermocycler and data computed with SDS Absolute Quantification Software Version 2.2.2 (Applied BioSystems).