Model 7900 genetic analyzer

Total RNA was extracted using the miRVana miRNA isolation kit (Ambion, #AM1560, Austin, TX, USA). RNA quality and concentrations were analyzed on a NanoDrop M-1000 spectrophotometer and an Agilent 2100 Bioanalyzer. RNAs with quality scores >7.00 were used for expression assays. RNA concentrations were standardized to 200 ng/μL. TaqMan low-density miRNA arrays (TLDAs) (Applied Biosystems, #4444913, Foster City, CA, USA) were used to assess miRNA expression levels in proliferating basal cells grown in SAGM-EA. Reverse transcription of 600 ng total RNA was carried out using a TaqMan miRNA reverse transcriptase kit (Applied Biosystems, #4366596) with Megaplex RT primers, Human Pool (Applied Biosystems, #4399966). Samples were loaded onto the TLDA, which utilizes 384 wells preloaded with specific miRNA probes and primers in each well. The TLDA data were processed on an Applied Biosystems Model 7900 Genetic Analyzer, and the data were analyzed using the Applied Biosystems StatMiner software. Each sample was analyzed in triplicate, and each Ct value was normalized to the Ct value of RNU48 endogenous RNA control. Relative quantification of each miRNA was performed using the ΔΔCt method. Statistical significance of the fold change was assessed using two-tailed t-tests. p-values of <0.05 were taken as statistically significant.

Total RNA was extracted from cultured cells using the miRvana miRNA Isolation kit (Ambion, Life Technologies). RNA yield and purity was assessed using a NanoDrop Model 1000 spectrophotometer (Thermo Scientific). Total RNA (500 ng) was oligo-dT reverse transcribed with SuperScript III (Invitrogen, Life Technologies). Real-time PCR was performed in triplicate on an Applied Biosystems Model 7900 Genetic Analyzer under standard conditions. Primer sequences are listed in S1 Table. Results were quantitated using the comparative cycle threshold (ΔΔCt) method [9 (link)]. Data were normalized to 18S rRNA.