Rt kits

Quantitative real-time PCR (qRT–PCR) was performed as previously described [10 (link)]. Briefly, total RNA was isolated using Tri-Reagent (Sigma Chemical Co., St. Louis, MO, USA) according to recommendations of the manufacturer. Total RNA (1 µg in 20 µL) was reverse transcribed using reverse transcription (RT) kits (Applied Biosystems; Foster City, CA, USA) following the manufacturer’s protocol. Primers for human RPLP1 variants 1(v1) and 2 (v2) as well as beta-actin (ACTB) were designed using Primer-Blast and synthesized by Integrated DNA Technology (IDT, Coralville, IA, USA): human RPLP1v1 (NM_001003): forward, 5′-TGACAGTCACGGAGGATAAGA-3′ and reverse, 5′-CCAGGCCAAAAAGGCTCAAC-3′; human RPLP1v2 (NM_213725): forward, 5′-CTCACTTCATCCGGCGACTA-3′ and reverse, 5′-GCCAGGGCCGTGACTGT-3′; human ACTB (NM_001101): forward 5′-GCACAGAGCCTCGCCTTT-3′; reverse, 5′-TATCATCATCCATGGTGAGCTGG-3′. The resulting material was then used for independent qRT–PCR. qRT–PCR was carried out on an Applied Biosystems HT7900 Sequence Detector. To account for differences in starting material, ACTB primers were used. All samples were run in triplicate and the average value was used in subsequent calculations. The 2-delta-delta CT method was used to calculate the fold-change values among samples as previously described [10 (link)].

Quantitative RT-PCR (qRT-PCR) was performed as previously described [23] (link), [24] (link). Briefly, total RNA was isolated using TRI-Reagent (Sigma Aldrich Chemical Company, St. Louis, MO) according to recommendations of the manufacturer. Total RNA (250 ng in 5 µl) was reverse transcribed using reverse transcription (RT) kits (Applied Biosystems; Foster City, CA) following the manufacturer's protocol with the following modifications. Briefly, miRNAs were reverse transcribed in a single reaction using 2 µl of each miRNA specific 5X RT primers. Resulting material was then used for independent qRT-PCR for miR-451 and miR-144 as well as fibrinogen alpha chain (Fga). RT-PCR was carried out on an Applied Biosystems HT7900 Sequence Detector. To account for differences in starting material, U6 was used for miR-451 and miR-144 while 18S was used for Fga. All primers were purchased from Applied Biosystems. Each cDNA sample was run in triplicate and the relative abundance of each target divided by the relative abundance of U6 for miRNAs and 18S for Fga in order to normalize for the starting quantity of cDNA. Each primer set included a minus template cDNA control. The delta-delta CT method was used to calculate the fold-change values among samples.