|
| Status |
Public on Sep 22, 2007 |
| Title |
PlagL2WT1 |
| Sample type |
RNA |
| |
|
| Source name |
mouse intestine pLagl2 wild type 1
|
| Organism |
Mus musculus |
| Characteristics |
18.5 dpc mouse small intestine wild type
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from wild-type mouse small intestines at 18.5 dpc using the Macherey-Nagel Nucleospin kit.
|
| Label |
biotin
|
| Label protocol |
RNA is converted into cDNA using a T7 promoter-tailed oligo-dT primer in the synthesis of the first cDNA strand; second strand cDNA synthesis is then carried out.
The double-stranded cDNA is used as the template in an in vitro transcription (IVT) reaction catalyzed by T7 polymerase and containing biotinylated CTP and UTP in addition to the four unmodified ribonucleoside triphosphates.
The biotinylated complementary RNA (cRNA) is purified from the IVT reaction mixture using the RNeasy system (Qiagen). The cRNA is quantified spectrophotometrically and purity of the cRNA is also assessed by spectrophometric measurements. Those cRNA.s that fall outside of an acceptable range will not be carried forward in the analysis. Should this occur, investigators would be notified and asked to provide a new RNA sample
|
| |
|
| Hybridization protocol |
The purified cRNA is fragmented in order to facilitate the subsequent hybridization step. The cRNA is purified from the fragmentation reaction using phenol/chloroform extraction and ethanol precipitation.
The fragmented cRNA is added to a hybridization solution containing several biotinylated control oligonucleotides (for quality control), and hybridized to a microarray chip overnight at 45°C.
The chips are then transferred to a fluidics instrument that performs washes to remove cRNA that has not hybridized to its complementary oligonucleotide probe. The bound cRNA is then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE); additional fluors are then added using biotinylated anti-streptavidin antibody and additional SAPE.
|
| Scan protocol |
Each cRNA bound at its complementary oligonucleotide is excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions are captured. These measures provide the basis of subsequent biostatistical analysis.
|
| Description |
none
|
| Data processing |
dChip
|
| |
|
| Submission date |
Sep 20, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
zhao chen |
| E-mail(s) |
zhao_chen@dfci.harvard.edu
|
| Phone |
617-582-7857
|
| Fax |
617-582-8490
|
| Organization name |
Dana Farber Cancer Institute
|
| Street address |
1 jimmy fund way
|
| City |
boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE9123 |
transcription factor PlagL2 regulates steps in chylomicron metabolism |
|
| Relations |
| Reanalyzed by |
GSE119085 |
