Kapataq extra hotstart readymix with dye

RNA isolation from differentiated PC12 cells was performed using a Blood/Cultured Cell Total RNA mini kit (Favorgen Biotech Corp., Taiwan). The extracted total RNA was subjected to reverse transcriptase reaction using a PrimeScript real-time PCR (RT-PCR) kit (TAKARA, Osaka, Japan). PCR was performed using KAPATaq EXtra HotStart ReadyMix with dye (KAPA Biosystems Inc., Woburn, MA, USA) and the following PCR primers: rat TrkA, 5′-ATG CTC GTC AGG ACT TCC ATC G-3′ and 5′-TAG CCA CAG CCA GAA GCT GC-3′; rat TrkB, 5′-AAG TCC TCT ATG AAG ACT GGA CC-3′ and 5′-TGC CAA ACT TGG AAT GTC TCG CCA-3′; rat TrkC, 5′-CAG CCC AGA GCC TTT GCT AAG-3′ and 5′-GGC AAA GGA GAG CCA GAG CCA TT-3′; rat p75NTR, 5′-CGG AAT TCG GAG ACA TGT TCC ACA GGC-3′ and 5′-CCT TGG GAT CCA TCG ACC-3′.

Hybridity was tested by RAPD-PCR with reference to Marubashi and Onosato¹⁶ and Tezuka et al.^{41 (link)}. Six PCR primers were used, including OPA-1, OPA-5, OPA-9, OPA-11, OPA-12, and OPA-15 (Operon Technologies). KAPA Taq Extra Hotstart Ready mix with dye (Kapa Biosystems) was used for PCR. The PCR reaction solution was prepared by combining 10 μL of 2 × KAPA Taq EXtra HotStart ReadyMix with dye, 2 μL of OPA primer at 10 μM, and 20 ng of DNA; the solution then was adjusted to 20 μL with PCR-grade water and mixed. The PCR reaction solution was subjected to initial denaturation at 95 °C for 15 min, followed by 45 cycles of denaturation at 94 °C for 1 min, annealing at 35 °C for 2 min, and extension at 72 °C for 3 min, using an Applied Biosystems 2720 Thermal Cycler (Life Technologies). The PCR products were electrophoresed (along with a commercial DNA size marker) on a 2% or 3% agarose gel supplemented with 0.01% GelRed (Biotium) using TAE buffer (4.84 g/L tris (hydroxymethyl) aminomethane, 1.14 mL/L acetate, and 370 mg/L EDTA) as the electrophoresis buffer, and the gel was photographed under UV light to detect the PCR products.

For reverse transcription (RT), we used RevertAid First Strand cDNA Synthesis Kit (Life Technologies) according to the manufacturer’s instructions. We used oligo-dT primers for the RT reaction. For semiquantitative PCR, we used KAPATaq Extra HotStart Ready Mix with dye (KAPA Biosystems) according to the manufacturer’s instructions.