|
| Status |
Public on Oct 04, 2017 |
| Title |
N27_Mn_rep3 |
| Sample type |
genomic |
| |
|
| Source name |
N27_Mn treated cells_rep3
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell line: N27
cell type: Rat Dopaminergic Neural Cell Line
treatment: Mn
|
| Treatment protocol |
Rat dopaminergic neural cells treated with treated Mn at sublethal dose.
|
| Growth protocol |
Rat dopaminergic neural cell line at standard condition in medium
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from cells of different experimental groups, subjected to proteinase K digestion in the presence of 1% SDS, followed by phenol: chloroform extraction, RNase A treatment and precipitation using NaCl and ethanol.
|
| Label |
Cy3
|
| Label protocol |
After hybridization at 42OC for 16 h, the slides were washed, blocked and then incubated in anti-5-methylcytosine antibody (1h at 25OC). Slides were then washed and incubated in cy3 labeled anti-mouse IgG (1h at 25OC)
|
| |
|
| Hybridization protocol |
Genomic DNA (2 µg) was sheared into 1.5-2 kb fragments by sonication and the fragments were denatured by heating at 95OC and hybridized on to a Rat GE 4x44K v3 Microarray slides (Agilent Technologies, CA, USA) each with 4x44K arrays, containing 44,000 probes of 22,583 genes.
|
| Scan protocol |
Scanned with ScanArray Express at 5 m resolution at 543 nm. Scanarray software was used for data acquisition, mean or median intensity calculation of each spot and normalization.
|
| Description |
Genome wide DNA methylation analysis of rat dopaminergic neural cell line treated with Mn _rep3
|
| Data processing |
For normalization of microarray data, LOWESS normalization was used as the default parameter for the array scanning apparatus. The microarray slides were provided with spots containing negative controls and positive controls, which provides a background signal. The fluorescence values from these spots were considered to be representatives of non-specific binding of the DNA to the slides and these values were used to determine the true positives in the data. These values were together termed as control values. The control values from each individual array were used to normalize the fluorescence for all the genes of that array in order to provide a uniform scale for comparison of genes between arrays. Assuming an error of 20 % in the ability to detect true positives, signals which were higher than 20% of the control value were considered a true positive. The normalization was performed by dividing the fluorescence values with the cutoff value of the respective arrays. For comparison of the genes between arrays the normalized values of fluorescence were used. The signal intensities were normalized considering signal to noise ratio of 1. This gave a quantifiable positive value in the form of fold intensity which was then compared within the replicates. All the spots which were identified as true positives in all three arrays were used for further analysis.
|
| |
|
| Submission date |
Dec 21, 2016 |
| Last update date |
Oct 04, 2017 |
| Contact name |
Santosh Chandrakant Narwade |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Pune
|
| Department |
Department of Zoology
|
| Lab |
Molecular Biology Research Laboratory
|
| Street address |
Ganeshkhind Road
|
| City |
Pune |
| State/province |
Maharashtra |
| ZIP/Postal code |
411007 |
| Country |
India |
| |
|
| Platform ID |
GPL14746 |
| Series (1) |
| GSE92691 |
Comparative analysis of 1-methyl-4-phenylpyridinium (MPP+) and manganese induced neurotoxic effects on DNA methylation in dopaminergic neurons |
|
