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Sample GSM5416585 Query DataSets for GSM5416585
Status Public on Dec 01, 2021
Title K5+SP 30' rep3
Sample type RNA
 
Source name CGNs_medium aspirated_K5 + 0% FCS + SP 200 nM, 0.5h, replicate 3
Organism Rattus norvegicus
Characteristics strain: Wistar
tissue: Cerebella
cell type: Cerebellar Granule Neurons (CGNs) primary culture
age: 8-day-old (P8)
Treatment protocol 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) after 0.5h switch from 25 mM (K25) to 5 mM KCl (K5) and treated with a maximal effective dose of SP (200 nM).
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.
 
Submission date Jul 03, 2021
Last update date Dec 01, 2021
Contact name Sebastiano Cavallaro
E-mail(s) sebastiano.cavallaro@cnr.it
Organization name IRIB-CNR
Street address Via Paolo Gaifami, 18
City Catania
ZIP/Postal code 95126
Country Italy
 
Platform ID GPL15084
Series (1)
GSE179383 Deciphering the conserved dynamic transcriptional signature and regulatory software governing neuronal fate commitment

Data table header descriptions
ID_REF
VALUE Normalized signal intensity

Data table
ID_REF VALUE
GE_BrightCorner 0.06255627
DarkCorner 0.15867853
A_44_P133119 -0.13576078
A_44_P1028743 -0.22359371
A_64_P085072 0.6206646
A_64_P215165 0.8625436
A_64_P030861 0
A_43_P16664 0
A_44_P367586 -0.055397272
A_44_P544340 -0.0905385
A_64_P100629 2.0166888
A_44_P248235 1.5927305
A_64_P134485 0.14703035
A_42_P581135 -0.044151306
ERCC-00085_231 0
ERCC-00034_158 0.049357414
A_42_P462015 0.15209246
A_43_P19012 0.04568672
A_44_P318957 -1.063252
ERCC-00076_93 0.06622791

Total number of rows: 30507

Table truncated, full table size 715 Kbytes.




Supplementary file Size Download File type/resource
GSM5416585_SG11360001_252827913177_S001_GE1_1105_Oct12_1_3.txt.gz 3.1 Mb (ftp)(http) TXT
Processed data included within Sample table

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