Taq polymerase

Small interfering (si)RNA for SETDB1 or FosB were designed and purchased from Bioneer Inc. (Daejeon, Korea). siRNA was transfected into A549 cells using lipofectamine 2000. After 48 hr post-transfection, total RNA was isolated using a TRIZOL kit (Duchefa, Haarlem, Netherlands). RNA sequence analysis was performed at BML Inc. (Korea). For RT-PCR experiment, complementary DNA (cDNA) was synthesized with hexamer from total RNA. Conventional PCR was performed as described in a previous study (12) (link). cDNA was mixed with specific primer sets in 0.2 mM dNTP, 1 U Taq polymerase, buffer containing 1.5 mM MgCl₂ (Enzynomics, Seoul, Korea). The primer sequences were SETDB1 S1 5’-TTA ACA CAG GCC CTG AAT TTC T-3’ SETDB1 AS1 5’-TAC CCC TGT GGG TAG ACA CTC T-3’, FosB S1 5’-TAC TCC ACA CCA GGC ATG AG-3, AS’ 5’-CTT CGT AGG GGA TCT TGC AG-3’, EGR2 S1 5’-ATT CTG AGG CCT CGC AAG TA-3’, AS’ 5’-TGC TTT TCC GCT CTT TCT GT-3’, actin S1 5’-GTG GGG CGC CCC AGG CAC CAG GGC-3’, and actin AS1 5’-CTC CTT AAT GTC ACG CAC GAT TTC-3’. PCR reactions were carried out in a Perkin Elmer Thermal Cycler 9600 (Applied Biosystems, MA, USA). PCR products were resolved in 1.5% agarose gels.

Conventional RT-PCR was performed in buffer solution containing template cDNA,
Taq polymerase (Enzynomics, Daejeon, Korea) and each primer. The following pairs
of primers were used: GnRHR forward 5’-ACCCACGCAGTACGGTATTC-3’;
reverse 5’-GTGGCTACACCACTGTCGAA-3’ and
β-actin forward 5’-AGCCATGTACGTAGCCATC-3’;
reverse 5’-ATCTTCATGGTGCTAGGAGC-3’ primers (Bioneer, Daejeon,
Korea). The optimum temperature cycling protocol was used as 95°C for 30
s, 60°C for 1 min and 72°C for 1 min, using the Gene Pro thermal
cycler (Bioer). The PCR products were run on a 2% agarose gel. The bands
were visualized using Dye Loading STAR(6X) (DYNE BIO, Seongnam, Korea) and
detected using Biospectrum Imaging System (Analytik Jena, Jena, Germany).