Stratagene mx3000p qpcr machine

RNA was isolated from V. vulnificus using the easy-BLUE™ total RNA extraction Kit (iNtRON Biotechnology, Seongnam, Korea) and treated with the RNase-free DNase set (Promega, Madison, WI, United States) to remove any residual DNA. The purified RNA was quantified using a Biophotometer (Eppendorf, Hamburg, Germany). Subsequently, cDNA was synthesized from 500 ng of RNA using the CellScript^™ All-in-One cDNA Master Mix (Cellsafe, Yongin, Korea) following the manufacturer’s instructions. One microliter of cDNA was used for RT-PCR analysis on a Stratagene Mx3000p qPCR machine (Agilent Technologies, Santa Clara, CA, United States) using QGreenBlue 2 × Green qPCR Master Mix (Cellsafe, Yongin, Korea). The RT-PCR reactions were performed in triplicate in a 96-well plate using primer shown in Supplementary Table S2. The PCR conditions used to amplify all genes were: 10 min at 95°C and 40 cycles of 95°C for 15 s and 64°C for 40 s. The genes encoding type I glyceraldehyde-3-phosphate dehydrogenase (RS_10395) and DNA-directed RNA polymerase subunit alpha (RS_13660) of V. vulnificus were used as endogenous loading controls. Quantification was carried out using the Light Cycle 480 II real-time PCR system software program.

Total RNA was extracted from 2 × 10⁶ Jurkat or SH-SY5Y cells using a standard Trizol protocol (Sigma-Aldrich). Isolated RNA was treated with a TURBO DNA-free™ Kit (ThermoFisher) according to the manufacturer’s procedure. cDNA was obtained by reverse transcription of 2 µg of RNA using a Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher) according to the manufacturer’s procedure. cDNA was used for the evaluation of HEXB, HEXA, GLB1, and TFEB gene expression by quantitative PCR (Q-PCR) in a Stratagene Mx3000P Q-PCR machine (Agilent Technologies) as previously reported [31 (link)]. Data were analyzed by the ΔΔCt method. The sequences of specific primers used in this work are listed in Table 1.

Total RNA was extracted from HaCaT keratinocytes or mouse skin using Tri-RNA Reagent (Favorgen, Ping-Tung, Taiwan). First-strand cDNA synthesis was performed with PrimeScript RT master mix (Takara, Shiga, Japan). The resulting cDNAs were subjected to real-time PCR using qPCR 2x Premix SYBR (Enzynomics, Daejeon, Korea) with a Stratagene Mx3000p qPCR machine (Agilent Technologies, CA, USA). PCR conditions used to amplify all genes were 10 min at 95 °C and 40 cycles of 95 °C for 15 s and 64 °C for 40 s. Expression data were calculated from the cycle threshold (Ct) value using the ΔCt method for quantification. RPLP0 was used for normalization. Oligonucleotides are listed in Supplementary Table S1.