cell type: Fetal Liver CD34+ progenitors 3 weeks after transfection with 7 factor (SOX2, OCT4, KLF4, MYC, LIN28, NANOG, SV40 T antigen) passage: p1 sample type: Fetal Liver CD34+ progenitors 3 weeks after transfection with 7 factor (SOX2, OCT4, KLF4, MYC, LIN28, NANOG, SV40 T antigen)
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared as described in the miRNeasy Mini Kit (Qiagen) with on-column DNase I digestion.
Label
Cy3
Label protocol
150 ng total RNA from each sample was labeled using the miRNA Labeling Reagent & Hybridization Kit (Agilent Technologies, Santa Clara, CA) as described in the instruction manual.
Hybridization protocol
All arrays were hybridized at 55ºC for 20 hours followed by wash procedure according to the miRNA Microarray System Protocol, Version 1.5, December 2007 (Agilent Technologies).
Scan protocol
Fluorescent signals were obtained by scanning with an Agilent scanner controlled by Agilent Scan Control 7.0 software.
Description
Individual sample
Data processing
Data were acquired with Agilent Feature Extraction 9.5.3.1 software for miRNA microarray. MicroRNA Expression data were extracted from Agilent miRNA Kits by Agilent Feature Extraction software and then Partek and Spotfire were employed for their normalization and analysis. The fluorescent signal data were imported into Partek where duplicate probe information was merged and were then normalized to the 75th percentile. The 75th percentile normalization process is similar to median normalization, above, except that each gene’s 75th percentile value is subtracted rather than its median. (Agilent recommends the 75th percentile based on the observation that about half the microRNAs are not expressed, so the 75th percentile is near the median for those that are).
