RT-PCR and qRT-PCR for hpd Gene Expression

For the reverse-transcription polymerase chain reaction (RT-PCR) and quantitative reverse-transcription PCR (qRT-PCR) of the hpd gene, total RNA was extracted from F. kingsejongi cells in the mid-exponential phase using the Hybrid-R™ RNA Purification Kit (GeneAll Biotechnology, Seoul, South Korea). A cDNA library from the total RNA sample was synthesized using the ReverTra™ Ace qPCR RT Kit (Toyobo, Osaka, Japan). The RT-PCR was conducted on a T100™ Thermal Cycler (Bio-Rad, Hercules, CA, USA). The tuf gene, encoding the translation elongation factor Tu, was served as a reference gene [41 (link)]. qRT-PCR was performed on a Rotor-Gene Q PCR machine (QIAGEN, Hilden, Germany) with SensiFAST™ SYBR^® No-ROX one-step kit (Bioline, Cincinnati, OH, USA). Quantification was carried out using the comparative Ct (2^{− Δ ΔCT}) method [42 (link)]. The primers used for RT- and qRT-PCR were Tuf_forward (5′-ATTCCAACAACTCAGCATCAT C-3′: the tuf gene), Tuf_reverse (5′-AGTATGAAACTGCTACCCGTC-3′: the tuf gene), Hpd_forward (5′-CTC CAATCAACGAGCACCTTA-3′: the hpd gene), and Hpd_reverse (5′- TCTTTGTAGCCC GGAAGAAAC-3′: the hpd gene).