Transcriptor rt reaction buffer

Knockdown of target genes using RNAi was basically preformed as described previously [24 (link)]. The siRNAs used here are listed in S2 Table. The RNAi efficiency was checked by real-time RT-qPCR with a set of primers listed in S2 Table. Total RNA (2 μg) extracted from the knockdown cells was treated with 2 U of RQ1 DNase (Promega) to remove genomic DNA in a 20 μl 1×Reaction Buffer (Promega) at 37°C for 30 min, followed by adding RQ1 DNase stop solution (Promega) and incubated at 65°C for 15 min. The DNase-treated total RNA (2 μg) was incubated at 65°C for 5 min in a 10 μL solution containing 2.5 μM oligo(dT18) primer, 60 μM random N6 primer, and 1 mM dNTPs, then cooled on ice. Subsequently, 10-μL mixture containing 2×Transcriptor RT reaction buffer (Roche), 10 U RNase inhibitor (Roche), and 5 U Transcriptor RTase (Roche) was added to the solution. The cDNAs were synthesized in the mixture by sequential incubation at 25°C for 10 min, at 55°C for 30 min, and at 85°C for 5 min. The PCR was performed in a 20 μL mixture containing a 1 μl aliquot of the cDNA solution, 0.2 μM of each PCR primers, and 1×KAPA SYBR FAST Master Mix optimized for LightCycler480(Kapa biosystems). The thermal cycling conditions included 45 cycles of 95°C for 10 s, 58°C for 20 s, and 72°C for 1 s. Amplification of cDNA was monitored by LightCycler 480 (Roche).

Total RNA was isolated from the MC3T3-E1 pre-osteoblasts using an Invitrogen RNA isolation kit (Invitrogen, Carlsbad, CA, USA). cDNA synthesis was performed using 0.5–1 μg total RNA in 20 μL reaction mix consisting of 5 units Transcriptor Reverse Transcriptases (Roche Diagnostics, Basel, Switzerland), 0.08 A₂₆₀ units of random primers (Roche Diagnostics), 1 mM of each dNTP (Invitrogen), and 1× concentrated Transcriptor RT reaction buffer (Roche Diagnostics). Real-time PCR (RT-PCR) reactions were performed using the LightCycler^® 480 SYBR green I Master reaction mix according to the manufacturer’s instructions (Roche Diagnostics) in a Light Cycler 480 (Roche Diagnostics), and relative housekeeping gene expression (PBGD) and relative target gene expression, such as runt-related transcription factor 2 (Runx2), osteocalcin (Ocn), fibroblast growth factor 2 (Fgf2), dentin matrix protein 1 (Dmp1), and osteopontin (Opn) were determined. The primers (Invitrogen) used for RT-PCR are listed in Table 1. The values of target gene expression were normalized for PBGD gene expression.