Thermo scientific μdrop plate

The total RNA of placenta tissue was extracted using Trizol (Invitrogen, Carlsbad, CA, USA), and quantified at an absorbance ratio of 260/280 with the Thermo Scientific™ μDrop™ Plate (Thermo Scientific, Waltham, MA, USA) and Multiscan GO (Thermo Scientific, Waltham, MA, USA). The 500 ng of total RNA was reverse transcribed to complementary DNA (cDNA) using the Improm-II Reverse transcription system (Promega, Fitchburg, MA, USA). Polymerase chain reaction (PCR) condition and selection of primers were conducted following the description by Hosseindoust et al. [54 (link)], and Gao et al. [55 (link)]. Reverse transcription-quantitative real-time PCR (RT-qPCR) was conducted using the Real-Time System (Mx3000P, Stratagen, La Jolla, CA, USA). The RT-qPCR primers including HSP70, GLUT1, GLUT3, GLUT4, and reference gene (β-actin) were designed (Table 5). The relative mRNA expression levels of β-actin as a housekeeping gene was used for normalizing gene expression. The relative fold change of mRNA was determined using 2−ΔΔCT method.

Total RNA was extracted from liver tissue using the nucleic acid extraction kit (AccuPrep, Universal RNA extraction kit, Bioneer), and quantified using with the Thermo Scientific™ μDrop™ Plate (Thermo Scientific, USA) and Multiscan GO (Thermo Scientific, USA) at an absorbance ratio of 260/280. Complementary DNA (cDNA) was synthesized using Accupower cyclescript RT premix (Bioneer, Korea) according to the manufacturer’s instructions. RT-qPCR was performed using the CFX Real-Time System (C1000 Thermal cycler, Bio-Rad, Hercules, CA, USA). The RT-qPCR primers including HSPs (HSP108, HSP90, HSP70, HSP60, HSP47, HSP40, HSP27), ST13 (suppression of tumorigenicity 13), HSFS (HSF1, HSF2, HSF3), TNF-α (tumor necrosis factor alpha), CRYAB (alpha crystallin B), IFNG (interferon gamma), IL-6, IL-1B, and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) were designed (Table 3). The cDNA amplification was performed using SsoFast EvaGreen Supermix (Bio-Rad) under the following conditions: an enzyme activation step at 95 °C for 2 min, followed by denaturation and annealing/extension steps involving 40 cycles each at 95 °C for 5 s and at 60 °C (or each primer’s annealing temperature) for 5 s. The gene expression was normalized with GAPDH which is a housekeeping gene. The related gene expression was calculated via 2^−ΔΔCT method [18 (link)].