4.8. RNA Isolation and Real-Time Reverse Transcription-Quantitative PCR (RT-qPCR) Analysis
Total RNA was isolated using TRIzol Reagent (Carlsbad, CA, USA), following manufacturer instructions. 500 ng of total RNA was subjected to cDNA synthesis using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Real-time PCR reactions were performed with SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) and oligos for human TGM2 (forward–5′-TGTGGCACCAAGTACCTGCTCA-3′ and reverse–5′-GCACCTTGATGAGGTTGGACTC-3′), LGR5 (forward–5′-CTGTCGTCTTTTCCTATAACTGGG-3′ and reverse–5′-GAATCTTATAGGCATTCTCACACAC-3′), ALPI (forward–5′-CCAAGTCCTTGGGTCTACCA-3′ and reverse–5′-TCCCCTCAGGTTGTTCTCTG-3′) and GAPDH (forward–5′-GTCTCCTCTGACTTCAACAGCG-3′ and reverse–5′-ACCACCCTGTTGCTGTAGCCAA-3′) in triplicate.Results presented are calculated as a log2 Fold change compared to the reference sample (WELR500), normalized by housekeeping gene expression (GAPDH) as described [