On day five in culture, neuronal cultures were incubated in C2 culture media only (control) for 24h or with 50 ng/ml bone morphogenic protein-7 (BMP7, Curis, Cambridge, MA) in C2 for 6 or 24 hours. The experimental design described herein resulted in three total conditions: (1) C2 media only for 24h (Control), (2) C2 +BMP 6h, and (3) C2 +BMP 24h. This treatment protocol was repeated three times for each of the three biological replicates included in the study.
Growth protocol
All procedures involving animals were performed according to protocols approved by the Institutional Animal Care and Use Committee at Johns Hopkins University. Superior cervical ganglia (SCG) were dissected from E21 Holtzmann rat pups. SCG were dissociated (Higgins, D, et al 1991) were plated on Collagen I-coated dishes in C2 culture media (serum-free medium) plus beta-nerve growth factor (100ng/ml, Harlan Bioproducts, Indianapolis, IN) as previously described. One day after plating, cells were treated with Ara-C (Sigma) for 48 hours to deplete cultures of non-neuronal cells including glial cells that would otherwise generate and excrete BMP. On day four, media was changed to C2 without Ara-C. The neuronal growth protocol was repeated three times to give three independent, biological replicates.
Extracted molecule
total RNA
Extraction protocol
Following incubation with or without BMP7 for the prescibed length of time (6 or 24h), superior cervical ganglia (SCG) neurons were dislodged from the cultured plate and pooled to yield a sufficient number of cells per treatment condition. Total RNA was isolated from each pooled sample according to the RNeasy Total RNA kit (Qiagen, Valencia, CA) manufacturer’s protocol. RNA concentration was determined by spectrophotometric absorbance at 260nm, and the 260nm to 280nm absorbance ratio was calculated to define RNA purity.
Label
biotin
Label protocol
Each total RNA sample was labeled and amplified following the recommended protocol in the Affymetrix GeneChip Expression Analysis Technical Manual, rev.3 (http://www.affymetrix.com/support/downloads/manuals/expression_analysis_technical_manual.pdf). Using x µg of total RNA as input for all samples, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (IDT, Coralville, IA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, the cDNA was converted to labeled cRNA (the target) using T7 RNA polymerase in the presence of biotinylated UTP and CTP (Enzo, Farmingdale, NY). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen), followed by an ethanol precipitation of the labeled target. The target was fragmented to produce a uniform distribution of short cRNAs. 10µg (microarray processing batch1) or 5ug (microarray processing batch2) of each of fragmented target was combined with hybridization control oligomer (Affymetrix, Santa Clara, CA) and control cRNAs (1.5pM BioB, 5.0pM BioC, 25pM BioD, and 100pM Cre; Affymetrix, Santa Clara, CA), in hybridization buffer and applied to the array.
Hybridization protocol
The arrays were hybridized in a GeneChip Hybridization Oven 640 (Affymetrix, Santa Clara, CA) for 16 hours at 45⁰C, followed by washing, staining with streptavidin-phycoerythrin (SAPE; Molecular Probes, Eugene, OR), signal amplification with biotinylated anti-streptavidin antibody (Vector Labs, Burlingame, CA), and a final staining with SAPE. The staining and washing were performed using GeneChip Fluidic Station 400s (Affymetrix, Santa Clara, CA) running standard Affymetrix fluidics script, EukGE_WSv5 450.
Scan protocol
The distribution of fluorescent material on the processed array was determined using the Affymetrix GeneChip Scanner 3000 with AutoLoader. The array image scan was processed using Affymetrix GeneChip Operating Software (GCOS) v. 1.2, which employs the Microarray Suite, version 5.0 (MAS 5.0) algorithm. Image processing resulted in the generation of raw, probe level data in the CEL file format.
The data were processed by Partek Genomic Suite (Partek, Inc, St. Louis, MO) software using Robust Multichip Analysis (RMA) that includes a background correction, quantile normalization, and median polish as summarization. The batch correction statistical algorithm was applied to minimize the technical variation observed between the two microarray processing batches, which differed by scan date and cRNA amount hybridized to the microarray.
