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Sample GSM375692 Query DataSets for GSM375692
Status Public on Dec 18, 2009
Title 2 months, ipsilateral group, biological replicate 1
Sample type RNA
 
Source name L4/L5 dorsal root ganglia from adult rats (2 months old) 7 days after SNI surgery
Organism Rattus norvegicus
Characteristics strain: Sprague-Dawley
tissue: Dorsal root ganglia
postnatal age: 2 months
surgery type: Spare nerve injury
experimetal group: Adult Ipsilateral
Treatment protocol Experiments were performed on adult (2 months old) male Sprague–Dawley rats and rat pups (10 days postnatal age, P10) and carried out in accordance with the United Kingdom Animal (Scientific Procedures) Act 1986. Spared nerve injury and sham surgery were performed under halothane general anaesthesia with antiseptic conditions. After skin preparation, the sciatic nerve and its three terminal branches were exposed in the upper lateral thigh. Axotomy and ligation of the common peroneal and tibial branches was performed under direct vision, leaving the sural nerve intact. Muscle and skin were closed in two layers. In the sham operation, the procedures were the same but the nerves were only exposed and not cut or ligated. In all cases great care was taken not to stretch the nerve, or its branches, nor to damage the intact nerves.
Growth protocol After the surgery, the animals were returned to their cages and litters and maintained on a 12 hour light/dark cycle at constant ambient temperature in the Biological Services Unit with free access to food and water for 7 days post surgery (ps) until DRGs collection.
Extracted molecule total RNA
Extraction protocol Seven days post surgery, animals were sacrificed with 0.1 mL of Euthanal®, the fresh DRGs were snap frozen on liquid nitrogen and stored at –80o C until RNA extraction. L4 and L5 DRGs ipsilateral to the lesion were collected and pooled from 3 animals per adult experimental group and 4 animals per P10 group, (i.e. n= 6 DRGs per adult sample , n=8 per P10 sample) to minimise inter-animal differences. RNA was extracted using Trizol® reagent (Invitrogen®) and QIAshredder column (Qiagen®) for homogenisation according to manufacturer’s protocols. Additionally, the RNA was cleaned by ”RNA purification column” (Qiagen®) and a final volume of 50 uL obtained according to manufacturer protocol. The amount of RNA was measured with a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Inc.) and its quality assessed with Experion RNA HighSens StdSens analysis kit (Bio-Rad®). Then, the RNA was precipitated and dissolved to obtain a final concentration of 1 µg/µL and stored at -80°C until for cRNA synthesis or amplification by quantitative real-time polymerase chain reaction (qPCR).
Label biotin
Label protocol cRNA synthesis First strand cDNA synthesis; 2 g (for qPCR) or 10 ug (microarray analysis) of total RNA and 100 pmol/uL of the T7(dT)24 primer (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’) were combined for annealing at 70o C for 10mins and chilled on ice for 2mins. Then, the SuperScript III kit (Invitrogen®) was added according to manufacturer indications to synthesize the cDNA. Second strand cDNA synthesis; the whole amount of the cDNA produced was used as template for the second strand synthesis plus 10 units of E. coli DNA ligase, 40 units of E. coli DNA polymerase (Invitrogen®), 3uL of 10 mM dNTPs and 2 units of Rnase H (Invitrogen®). The reaction was stopped by addition of 10 uL of 0.5M EDTA. The double stranded cDNA was then purified and precipitated and the resuspended in 10uL of nuclease free water. Synthesis of biotin-labelled cRNA; we used the Enzo-Bioarray kit (Enzo Life Sciences, Inc) for synthesis of the cRNA according to manufacturer protocol. The resulting cRNA was purified with Rneasy kit (Qiagen®). The concentration was measured on a spectrophotometer and 20 µg of each sample was fragmented in 200 mM tris-acetate (pH= 8.1), 500mM potassium acetate and 150 mM magnesium acetate for 35mins at 95°C, then chilled on ice and stored at -80°C until microarray hybridisation
 
Hybridization protocol For the mRNA high-throughput screening we used the GeneChip ® Rat expression 230 version 2.0 arrays and the GeneChip® Hybridization, Wash and stain kit from Affymetrix®. The protocol of hybridisation, according to manufactured indications was the 49/64 formats, which corresponds to the selected type of array. The control oligonucleotides were: B2 and the Eukaryotic bioB, bioC, bioD and cre. Then, the samples were washed and stained on the Fluidics Station 450 (Affymetrix®) monitored with the GeneChip® Operating Software (Affymetrix®) according to manufactured instructions (using the protocol FS450_003 which corresponds to rat 230 v2.0 array).
Scan protocol We used GeneChip Scanner 3000 (Affymetrix®).
Description The L4/L5 dorsal root ganglia in the ipsilateral group are those ganglia ipsilateral to the surgery in the spare nerve injury group
Data processing The data were analyzed with 'R software' loaded with the 'Bioconductor' platform using GC-RMA normalisation and Limma test to contrast the experimental groups.
 
Submission date Feb 27, 2009
Last update date Dec 18, 2009
Contact name David Vega-Avelaira
E-mail(s) david.vega74@gmail.com
Organization name University College of London
Street address Gower street
City Lonndon
ZIP/Postal code wc1e 6bt
Country United Kingdom
 
Platform ID GPL1355
Series (1)
GSE15041 Postnatal developmental changes in Sprague-Dawley rats in the model of neuropathic pain 'spare nerve injury'

Data table header descriptions
ID_REF
VALUE GC-RMA

Data table
ID_REF VALUE
1367452_at 12.15
1367453_at 10.52
1367454_at 10.36
1367455_at 12.95
1367456_at 11.92
1367457_at 11.01
1367458_at 8.5
1367459_at 12.96
1367460_at 13.39
1367461_at 10.55
1367462_at 12.97
1367463_at 10.22
1367464_at 11.17
1367465_at 11.33
1367466_at 10.04
1367467_at 13.15
1367468_at 8.49
1367469_at 13.81
1367470_at 11.12
1367471_at 9.66

Total number of rows: 31099

Table truncated, full table size 489 Kbytes.




Supplementary file Size Download File type/resource
GSM375692.CEL.gz 2.8 Mb (ftp)(http) CEL
Processed data included within Sample table

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