subject status: healthy donor tissue: Oesophageal squamous mucosa percentage of barrett's at 3-5 years: N/A molecule subtype: microRNA
Treatment protocol
Musocal biopsy samples were collected from patients with Barrett's oesophagus or healthy controls. After collection, they were immediately stored in RNAlater® solution (Ambion, Austin, Texas, USA) as the manufacturer’s protocol indicated
Extracted molecule
total RNA
Extraction protocol
After the biopsies were thawed and extra RNAlater@ solution removed, the samples were homogenized in 500 ul TRIzol@ Reagent (Invitrogen, Carlsbad, CA, USA) and the manufacturer's protocol was followed to extract RNA. The RNA concentration was determined using the Biophotometer.
Label
FAMTM
Label protocol
PCR assays were performed by following the protocol for TaqMan@OpenArray Human MicroRNA Panel (4461104, Life technologies). 45 ng of RNA was used in the reverse transciption reaction for each sample. Megaplex Primer pools, Human Pools set v3.0 was used in the assay.
Hybridization protocol
n/a
Scan protocol
n/a
Data processing
Raw fluorescence data was processed using OpenArray® Real-Time qPCR Analysis Software (BioTrove™, version 1.0.4). Raw fluorescence data was exported in .csv file format for subsequent processing in R software. The CSV files were imported into R software (3.1.0) and the fractional cycle (Ct) at which the second derivative maximum of a logistic curve fit to the raw fluorescence data was determined. Any microRNA that had no Ct value for a particular sample was assigned the value of 40. Mann Whitney U tests were used to generate lists of differentially expressed microRNAs in the comparison of control squamous tissues vs post-ablation neosquamous tissues, and for the comparison of post-ablation squamous tissues vs. post-ablation neosquamous tissues. Significance was set at p<0.05. False Discovery rates were estimated using the Benjamini-Hochberg procedure (Benjamini, Yoav; Hochberg, Yosef (1995). Controlling the false discovery rate: a practical and powerful approach to multiple testing (PDF). Journal of the Royal Statistical Society, Series B. 57 (1): 289–300.) Fold Change worksheet reports the the ratios of normalized relative expression levels.
