Journal: Archives of virology

Article Title: In-depth serum virome analysis in patients with acute liver failure with indeterminate etiology

doi: 10.1007/s00705-019-04466-9

Figure Lengend Snippet: Confirmation of a bacterium-like sequence (alf1_3) originating from a reagent. The contig, which was unique to ALF case 1, was examined by nested PCR using 5 μL of template from multiple sources, including total RNA extracted from ALF cases 1 and 2 (lanes 1 and 2), SuperScript III reverse transcriptase (lanes 3 and 4), 5x SuperScript III reverse transcriptase buffer (lanes 5 and 6), DTT (lanes 7 and 8), RNaseOUT Recombinant Ribonuclease Inhibitor (lanes 9 and 10), RNasin Ribonuclease Inhibitors (Promega) (lanes 11 and 12), and a negative control (H2O, lane 13) (A). Sanger sequencing of the PCR amplicon showed that its sequence was identical to that of the contig (B). M, 1-kb DNA ladder (Invitrogen).

Article Snippet: In brief, 10 μL of total RNA was used for 20-μL RT consisting of 1x SuperScript III reverse transcriptase buffer, 10 mM DTT, 1 μM reverse primer R1 (5′- TCG GCA ACA ACA AGA CCA TC - 3′), 1 mM dNTPs (Invitrogen), 16 U of RNasin Ribonuclease Inhibitor (Promega), and 200 U of SuperScript III reverse transcriptase (Invitrogen).

Techniques: Sequencing, Nested PCR, Reverse Transcription, Recombinant, Negative Control, Amplification