Nanodropnd 1000

The samples were lysed using TRIzol reagent (Lifetech 15596026), and the concentration of RNA in DEPC in the kit was measured with a UV spectrophotometer (DeNovix or NanoDropND-1000). The volume of total RNA required for reverse transcription was calculated, and the first-strand cDNA was synthesized according to the instructions of the FastKing RT Kit (With gDNase). After target gene was amplified by a quantitative PCR instrument (TL988 or LightCycle 96), Ct value analysis was performed. In this study, the q-PCR primers were designed using Beacon Designer 7.90, and the primer sequences are shown in Additional file 9: Table S2 (F: stands for upstream primer, FORWARD; R: downstream primer, REVERSE).

RT-PCR was conducted to evaluate expression levels of mRNA for the caspase-3 gene. Total RNA was isolated from liver tissues homogenized with Trizol reagent (Invitrogen, USA) and purified using the easy spin RNA extraction kit (Intron Biotechnology, India). RNA quality and concentrations were determined using a NanoDrop ND-1000 (NanoDrop DS-11 FX; DeNovix, Delaware, USA). Reverse transcription and real-time quantitative PCR (qPCR)(QuantStudio-1 Real time PCR system, Applied Biosystem, Thermofisher Scientific USA)) were carried out on RNA samples for caspase-3 and GAPDH (as a housekeeping gene) using one-step RT qPCR kit (SYBR Green with low ROX, Intron biotechnology-Korea). The primers used for Caspase-3 and GAPDH genes expression are shown in Table 1. For gene expression quantification, the comparative threshold cycle (ΔΔCt) method was used following Applied Biosystems/Life Technologies’ guidelines. Results were normalized to GAPDH expression and expressed as arbitrary units.