Pcrmax alpha thermal cycler

The intestine samples were homogenized using an ultraturax homogenizer (D1000 Handheld homogenizer, Benchmark Scientific Inc., Sayreville, NJ, USA). The total RNA was isolated using TRIzol reagents according to the Direct-zol RNA Miniprep (R2052, Zymo Research Orange, CA, USA) kit protocol. The quantity and purity of RNA were determined in the microplate reader (Synergy HT Multi-Mode Microplate Reader-SN 1712214, BioTek Instruments, Inc., Winooski, VT, USA). The absorbance was read with the help of a Gen5 microplate and software (BioTek version 3.03). The quality and integrity of RNA were checked by Qubit RNA IQ assay kit (#Q33222, Thermo Fisher Scientific) using a Qubit 4 fluorometer (Invitrogen by Thermo Fisher Scientific). The RNA IQ number (which indicates the RNA sample integrity and quality) ranged from 8.7 to 10. The total RNA was used directly to synthesize cDNA. The cDNA synthesis was done using LunaScript RT Super Mix Kit (Cat. No. E3010L, New England Biolabs, Inc., USA). In brief, the cDNA synthesis was made using 200 ng of total RNA through the reaction process of 2 min at 25 °C, 10 min at 55 °C and lastly, 1 min at 95 °C by PCR^max Alpha Thermal Cycler (Cole–Parmer Ltd., UK). The cDNA was stored at −80 °C until RT-PCR assay.