cell type: BMSC manufacturing method: Alpha MEM with UltraGlutamine1 20% FBS┼ center: #6 lot: B
Extracted molecule
total RNA
Extraction protocol
Total RNA from BMSC samples were isolated and purified by using miRNeasyKit (Qiagen, Germantown, MD, USA). The RNA concentration was determined using Nano Drop ND-1000 Spectrophotometer (Nano Drop Technologies, Wilmington, DE, USA) and RNA quality was assessed with Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Label
Cy5
Label protocol
RNA was amplified and labeled using an Agilent LowInput QuickAmp Labeling Kit and subsequently mixed with Universal Human Reference RNA (Stratagene, Santa Clara, CA, USA)
Total RNA from BMSC samples were isolated and purified by using miRNeasyKit (Qiagen, Germantown, MD, USA). The RNA concentration was determined using Nano Drop ND-1000 Spectrophotometer (Nano Drop Technologies, Wilmington, DE, USA) and RNA quality was assessed with Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Label
Cy3
Label protocol
RNA was amplified and labeled using an Agilent LowInput QuickAmp Labeling Kit and subsequently mixed with Universal Human Reference RNA (Stratagene, Santa Clara, CA, USA)
Hybridization protocol
RNA was amplified and labeled using an Agilent LowInput QuickAmp Labeling Kit and subsequently mixed with Universal Human Reference RNA (Stratagene, Santa Clara, CA, USA) and cohybridized to Agilent Chip Whole Human genome, 4x44k slides according to the protocol provided by Agilent.
Scan protocol
The slides were incubated for 17h at 65°C and then the scanned using the Agilent B Scanner.
Data processing
Raw images were obtained by scanning the slides with an Agilent Scan G2505B and Agilent Scan Control software (version 9.5). The images were extracted using the Feature Extraction Software (Agilent Technologies). Partek Genomic Suite 6.4 (Partek Inc., St. Louis, MO, USA) was used for data visualization, identification of differentially expressed transcripts and hierarchical cluster analysis. We transformed the fluorescence intensity data to log2 ratios of each sample versus the universal human RNA reference (Stratagene, Santa Clara, CA, USA).
Manufacturing Differences Affect Human Bone Marrow Stromal Cell Characteristics and Function: Comparison of Production Methods and Products from Multiple Centers
