normalization data transformation protocol
Image analysis was carried out using the ImaGene 8.0 software (BioDiscovery, Inc.,USA). The quantified signals were background corrected (Normexp with offset value 10 according to the convolution method described by Ritchie ME, Bioinformatics (2007), Vol. 23, no. 20, pages 2700-2707. The signals were further normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm. ID_REF = VALUE = normalized log2 ratio (Hy3/Hy5) representing test/reference
array scanning protocol
Scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technoliges, Inc., USA) in an ozone-free environment
Hy3™-labeled immunoprecipitated RNA and Hy5™-labeled reference RNA were mixed pair-wise and hybridized to the miRCURY™ LNA array version 11.0 according to the miRCURY™ LNA array manual using a Tecan HS4800 hybridization station. Hybridized slides were stored in an ozone-free environment (ozone levels below 2.0 ppb)
The miRCURY™ LNA Array power labelling kit was used to label 600 - 1000ng of RNA isolated from immunoprecipitation with Hy3 and reference samples from total RNA with Hy5.
sample treatment protocol
Proliferating C2C12 muscle cells were grown in DMEM supplemented with 20% FBS and 1% Pen/Strep. At 80% confluency, cells were collected.
nucleic acid extraction protocol
RNA was extracted with phenol/chloroform from proliferating C2C12 cell extracts that were subject to immunoprecipitation with an antibody that recognized either HuR or IgG. Total RNA was isolated from proliferating C2C12 using TRIzol reagent and used as reference sample.