Invitrogen purelink rna mini kit

Following GSIS testing, cells were lysed for gene analysis. Nuclear mRNA was extracted and purified using the Invitrogen PureLink RNA Mini Kit (ThermoFisher Scientific, ON, Canada) protocol and evaluated by NanoDrop quantification. Purified RNA samples were stored at −80°C until gene expression analysis using the CFX Opus Real-Time PCR system (BioRad, ON, Canada). Primers specific to beta-cell genes were chosen to evaluate the spheroids in terms of insulin production and beta-cell maturation. Forward and reverse sequences for reference and target genes were obtained from Integrated DNA Technologies (IA, USA) and prepared for testing using the Luna OneStep reaction kit (New England Biolabs, MA, USA). mRNA was reverse transcribed into cDNA, amplified by thermal cycling, and detected by SYBR green fluorescence. Target gene expression was quantified relative to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) within each group and normalized to the monoculture spheroid control group for fold change analysis (R = 2^−ddCt). Glut2 is a transmembrane glucose transporter, and PDX1 is involved with endoplasmic reticulum health and function, while MAFA is a beta-cell specific transcriptional activator (primer sequences and gene descriptions shown in table S2, visual diagram in figure S4) [30 (link)].

Tissues or cells were lysed in TRIzol, and RNA was isolated using an Invitrogen™ PureLink RNA Mini Kit (Cat No: 12183025, ThermoFisher) according to the manufacturer’s instructions. RNA concentration was determined by NanoOne, and an equal amount of RNA was used to prepare cDNA using LunaScript® RT SuperMix (New England BioLabs, Cat no: M3010L) according to the manufacturer’s conditions. qPCR was performed by TaqMan primer probes using Luna Universal Probe qPCR Master Mix (New England Biolabs, Cat No: M3004) and Bio-Rad i-cycler according to standard lab procedures^{9 (link),10 (link),31 (link)}. HPRT was used to normalize gene expression^{31 (link),32 (link)}. The ∆∆Ct method was used to calculate relative gene expression values in qPCR analysis.

Hepatic samples were homogenized in TRIzol reagent and total RNA was extracted using Invitrogen PureLink RNA Mini Kit (ThermoFisher, Waltham, MA, USA). RNA concentration and purity were determined by a Biodrop Touch Duo spectrophotometer (Montreal Biotech Inc., Dorval, QC, Canada) [31 (link)]. Complementary DNA was obtained by reverse transcripting 1 μg of RNA with the Superscript VILO Master Mix (Invitrogen, Waltham, MA, USA). Gene expression was analyzed by quantitative RT-PCR using the 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The thermal profile included an initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 3s, and annealing and extension at 60 °C for 30 s. Amplified gene expressions were quantified by fluorescence using the PowerUp SYBR Green Master Mix (Life Technologies, Carlsbad, CA, USA) [31 (link)]. Levels of expression of target-gene mRNAs were calculated by the 2^−∆∆CT method. The list of all primers can be found in the Supplementary Materials (Table S1).