|
| Status |
Public on Jan 27, 2011 |
| Title |
Gfp(1) |
| Sample type |
RNA |
| |
|
| Source name |
Cultured hippocampal neurons lysate
|
| Organism |
Mus musculus |
| Characteristics |
strain: CD1
cell type: hippocampal neuron
div: 5
genotype/variation: control
|
| Treatment protocol |
Neurons were transduced at 5 DIV using Sindbis virus bearing myc-tagged Ngn3 or GFP as control. After 1h viral particles were removed and proteins were allowed to express during 16h.
|
| Growth protocol |
Neurons were plated on 6-wells plates coated with poly-L-lysine at a density of 150–300 neurons/mm2, and they were cultured in Neurobasal supplemented with B-27 and GlutaMAX I.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lysed and total RNA was extracted using illustra RNAspin Mini RNA isolation kit from GE Healthcare. RNA quality was analyzed using a BioAnalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Biotin
|
| Label protocol |
The biotin-labelled cRNA target is prepared by a linear amplification method. The poly(A)+ RNA is primed for reverse transcription by a DNA oligonucleotide containing the T7 RNA polymerase promoter 5’ to a d(T)24 sequence. After second-strand cDNA synthesis, the cDNA serves as the template for an in vitro transcription (IVT) reaction to produce the target cRNA. The IVT is performed in the presence of biotinylated nucleotides to label the target cRNA.
|
| |
|
| Hybridization protocol |
Hybridization is performed from 10 μg of cRNA, overnight and at 37ºC in a shaking incubator, according to array manufacturers. Post-hybridization processing includes a stringent wash to remove unbound and nonspecifically hybridized target molecules, a staining step with a Cy™5-Streptavidin conjugate, and several non-stringent washing steps to remove unbound conjugate.
|
| Scan protocol |
Hybridized arrays were scanned on an Agilent Microarray Scanner (G2565BA, Agilent Technologies)
|
| Data processing |
CodeLink Expression Analysis software was used for primary data extraction from bioarray images. Microarray data were analyzed using the R language and packages from the Bioconductor project (http://www.bioconductor.org/). The codelink (Diez et al, 2007) package was used for preprocessing the arrays, genefilter (Gentleman et al, 2007) for data filtering and limma (Smyth, 2004) for statistical analysis. For preprocessing, background was corrected using the normexp method and quantile normalization was performed
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| |
|
| Submission date |
Jan 27, 2011 |
| Last update date |
Jan 27, 2011 |
| Contact name |
Ana Dopazo |
| E-mail(s) |
adopazo@cnic.es
|
| Phone |
34914531217
|
| Organization name |
CNIC
|
| Department |
Genomics Unit
|
| Street address |
Melchor Fdez Almagro
|
| City |
madrid |
| ZIP/Postal code |
28029 |
| Country |
Spain |
| |
|
| Platform ID |
GPL8063 |
| Series (1) |
| GSE26911 |
Comparative gene expression profile of Ngn3-overexpressing cultured hippocampal neurons vs the corresponding control populations |
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