Transcriptor reverse transcriptase reaction buffer

Total RNA was isolated from the MC3T3-E1 pre-osteoblasts seeded at 1 × 10⁵ cells/disc coated with OCP without/with κ-carrageenan after 1, 14, and 21 days of culture using an Invitrogen RNA isolation kit (Invitrogen). 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), 1 mM of each dNTP (Invitrogen), 0.08 A₂₆₀ units random primers (Roche Diagnostics), and 1x concentrated Transcriptor Reverse Transcriptase 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, i.e. runt-related transcription factor 2 (Runx2), osteocalcin (Ocn), fibroblast growth factor 2 (Fgf2), dentin matrix protein 1 (Dmp1), and osteopontin (Opn) were determined. Primers (Invitrogen) used for RT-PCR are listed in Table 1. Values of target gene expression were normalized for PBGD gene expression.

Primer-specific reverse transcription was performed using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). The reverse primers for partial E6 and E7 reverse transcripts were designed and utilized: CPV9E6mRNA-R: 5′-CCAGGCAGTCTGTACACCTC-3′; and CPV9E7mRNA-R: 5′-CAATCCTGATTCCACGACCG-3′. The internal control for dog ribosomal protein S5 (RPS5) was also included: RPS5-R: 5′-CCTGATTCACACGGCGTAG-3′ [34 (link)].
For each reaction, 1 µg total RNA, 1 µL 10mM reverse primer, and a variable volume of RNase-free water were prepared for a total reaction volume of 13 µL. Then, the mixtures were heated at 65 °C for 10 min to denature the secondary structure of the RNA and put on ice immediately after heating. Five microliters of Transcriptor Reverse Transcriptase Reaction Buffer (Roche), 0.5 µL Protector RNase Inhibitor (Roche), 2 µL 10 mM deoxynucleotide mix (Roche), and 0.5 µL Transcriptor Reverse Transcriptase (Roche) were directly added into each mixture. Following a short incubation at 55 °C for 30 min and 85 °C for 5 min, the primer-specific cDNA fragments were synthesized.