K5 neurons were also treated with a maximal effective dose of SP (200 nM), Pacap (100 nM) or Igf1 (3.26 pM). All the substances were obtained from Sigma-Aldrich (Milano, Italy), unless otherwise specified.
Growth protocol
Primary cultures of CGNs were prepared from 8-day-old Wistar rats (Charles River, Calco, Italy) and were cultured as previously described (ins ref). In brief, cerebella were sliced and tissue was dissociated through trypsinization in 0.025% trypsin solution (15 min at 37 °C) and trituration in presence of DNase (0.01%) and trypsin inhibitor (0.05%). Dissociated cells were collected through centrifugation and resuspended in basal Eagle’s medium supplemented with 10% fetal calf serum, 25 mm KCl, 2 mm glutamine and 100 μg/ml gentamycin. Cytosine arabinofuranoside (10 μM) was added after 18 h of culture to inhibit the growth of non-neuronal cells. After 6 days ‘in vitro’, extracellular KCl was shifted from 25 to 5 mm for neuronal apoptotic death induction. After two washes with serum-free BME containing 5 mm KCl, neurons were incubated with the same medium up to 48 h (K5), whereas control neurons were incubated with a serum-free medium supplemented with 25 mm KCl (K25).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with Trizol (Life Technologies, Monza, Italy) from four biological replicates for each experimental condition (K25, K5, K5+SP, K5+Pacap, K5+Igf1). RNA quantity and quality were assessed using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, MA, USA) and a 2100 Bioanalyzer microfluidic electrophoresis platform (Agilent Technologies, Palo Alto, CA), respectively, according to the manufacturer's instructions. Complementary RNAs (cRNAs) labeled with Cy3-CTP were synthesized from 1 μg of total RNA of each sample using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Palo Alto, CA), following the manufacturer's protocol.
Label
Cy3
Label protocol
Aliquots (750 ng) of Cy3‐labeled cRNA targets were hybridized on arrays using the SurePrint G3 Rat Gene Expression 8x60K microarray kit (Agilent Technologies, Palo Alto, CA).
Hybridization protocol
Microarray hybridization and washing were performed using reagents and instruments (hybridization chambers and rotating oven) as indicated by the manufacturer (Agilent Technologies, Palo Alto, CA).
Scan protocol
Arrays were then scanned at 3 µm resolution using an Agilent G4900DA SureScan Microarray Scanner System (Agilent Technologies, Palo Alto, CA).
Description
Gene expression in serum-deprived cerebellar granule neurons (CGNs) at 3h following incubation with a serum-free medium supplemented with 25 mm KCl (K25).
Data processing
Raw microarray data were acquired and analyzed using Agilent’s Feature Extraction v.12.1 software to assess the array spot quality, as well as to check signal and background intensity statistics in the default setting. Raw signal values were thresholded to 1, log2 transformed, normalized to the 75th percentile, and baselined to the median of all samples using GeneSpringGX v.14.9 (Agilent Technologies). Then, to identify genes with significant temporal expression changes and evaluate trend differences in CGNs undergoing apoptosis (K5 vs K25) and exposed to SP, PACAP or IGF1 treatment with respect to untreated (K5), pre-processed normalized array data were analyzed by using the R package maSigPro (microarray Significant Profiles). Genes whose expression levels varied in a statistically significant way along time were detected using the linear step-up Benjamini-Hochberg false discovery rate (FDR) procedure setting a corrected p-value ≤ 0.05.
