|
| Status |
Public on Apr 01, 2012 |
| Title |
N2 vs. CB4856, T3, rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
N2, 214 hours
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
genotype: wild type
age: 214 hours
developmental stage: old (senescent)
temperature: 24ºC
|
| Treatment protocol |
Reproductive nematodes from N2 and CB4856 were bleached (0.5 M NaOH, 1% hypochlorite) in order to collect age-synchronized eggs which were inoculated in 24 dishes (t0). After 40 hours (H1), nematodes from 6 dishes were collected in late L3 stage, frozen in liquid nitrogen and kept at -80ºC until the RNA extraction procedure. The remaining 18 dishes were kept in culture until hour 41 when the nematodes were transferred to fresh NGM dishes (with E. coli OP50) treated with 0.05-0.01 mg/ml of FUDR in order to avoid reproduction. After 30 hours in dishes with FUDR and when the reproductive period was about to finish, nematodes were transferred to fresh dishes without FUDR to prevent starvation. After 23 hours, 96 hours of total culture time (H2), nematodes from 6 dishes were collected and frozen in liquid nitrogen prior to RNA extraction. The remaining 12 dishes were kept at constant temperature until 214 hours of culture (H3), when they were harvested and frozen in liquid nitrogen. All the dishes were visually inspected before the harvest. Any population with contamination, more than one generation (reproduction) or starving nematodes (lacking bacteria) were discarded.
|
| Growth protocol |
Two C. elegans wild types, N2 and CB4856, were used to generate gene expression data at three different ages. The nematodes were cultured on standard nematode growth medium (NGM) with E. coli OP50 as food source and a constant temperature of 24ºC. Populations were started with nonmated hermaphrodites and screened regularly to remove any occurring males.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from nematodes was extracted following the Trizol method, followed by the RNeasy Micro kit (Quiagen, Valencia, CA, USA) to clean up the samples.
|
| Label |
Cy3
|
| Label protocol |
Labeled cDNA was produced with the Array 900 HS kit from Genisphere and Superscript II from Invitrogen. The Nucleospin kit was used to clean the cDNA samples in order to reduce unspecific binding to the arrays.
|
| |
|
| Channel 2 |
| Source name |
CB4856, 214 hours
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: CB4856
genotype: wild type
age: 214 hours
developmental stage: old (senescent)
temperature: 24ºC
|
| Treatment protocol |
Reproductive nematodes from N2 and CB4856 were bleached (0.5 M NaOH, 1% hypochlorite) in order to collect age-synchronized eggs which were inoculated in 24 dishes (t0). After 40 hours (H1), nematodes from 6 dishes were collected in late L3 stage, frozen in liquid nitrogen and kept at -80ºC until the RNA extraction procedure. The remaining 18 dishes were kept in culture until hour 41 when the nematodes were transferred to fresh NGM dishes (with E. coli OP50) treated with 0.05-0.01 mg/ml of FUDR in order to avoid reproduction. After 30 hours in dishes with FUDR and when the reproductive period was about to finish, nematodes were transferred to fresh dishes without FUDR to prevent starvation. After 23 hours, 96 hours of total culture time (H2), nematodes from 6 dishes were collected and frozen in liquid nitrogen prior to RNA extraction. The remaining 12 dishes were kept at constant temperature until 214 hours of culture (H3), when they were harvested and frozen in liquid nitrogen. All the dishes were visually inspected before the harvest. Any population with contamination, more than one generation (reproduction) or starving nematodes (lacking bacteria) were discarded.
|
| Growth protocol |
Two C. elegans wild types, N2 and CB4856, were used to generate gene expression data at three different ages. The nematodes were cultured on standard nematode growth medium (NGM) with E. coli OP50 as food source and a constant temperature of 24ºC. Populations were started with nonmated hermaphrodites and screened regularly to remove any occurring males.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from nematodes was extracted following the Trizol method, followed by the RNeasy Micro kit (Quiagen, Valencia, CA, USA) to clean up the samples.
|
| Label |
Cy5
|
| Label protocol |
Labeled cDNA was produced with the Array 900 HS kit from Genisphere and Superscript II from Invitrogen. The Nucleospin kit was used to clean the cDNA samples in order to reduce unspecific binding to the arrays.
|
| |
|
| |
| Hybridization protocol |
Microarrays were hybridized following the Genisphere Array 900 HS protocol with modifications.
|
| Scan protocol |
A Perkin Elmer scanner was used to extract the raw intensities.
|
| Description |
N2vsCBH3_rep3
|
| Data processing |
Preprocessing and normalization was done in the R software (http://www.r-project.org/) using Limma package. Loess method was used for normalization within arrays and normalization between arrays was done using aquantile method, both of them included in limma package.
|
| |
|
| Submission date |
Jul 12, 2010 |
| Last update date |
Apr 01, 2012 |
| Contact name |
Ana Viñuela |
| E-mail(s) |
ana.vinuela_rodriguez@kcl.ac.uk
|
| Phone |
+44 (0) 20 7188 1505
|
| URL |
http://www.anavinuela.com
|
| Organization name |
King's College London
|
| Department |
Twin Research & Genetic Epidemiology
|
| Street address |
St.Thomas' Hospital Campus Westminster Bridge Road
|
| City |
London |
| State/province |
London |
| ZIP/Postal code |
SE1 7EH |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL4038 |
| Series (1) |
| GSE22887 |
Dynamic gene expression of two Caenorhabditis elegans wild type strains (N2 and CB4856) |
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