|
| Status |
Public on Jan 08, 2007 |
| Title |
Expression analysis of primary mouse megakaryocyte differentiation-MK3 replicate |
| Sample type |
RNA |
| |
|
| Source name |
Mouse primary Megakaryocytes stage MK3
|
| Organism |
Mus musculus |
| Characteristics |
mouse CD1 day 14.5 fetal liver cultured megakaryocytes collected at day 3
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol reagents
|
| 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 |
Affymetrix MAS 5.0
|
| |
|
| Submission date |
Dec 21, 2006 |
| Last update date |
Jan 08, 2007 |
| Contact name |
Ramesh A Shivdasani |
| E-mail(s) |
ramesh_shivdasani@dfci.harvard.edu
|
| Phone |
617 632-5746
|
| Fax |
617 632-5417
|
| URL |
http://research.dfci.harvard.edu/shivdasani
|
| Organization name |
Dana-Farber Cancer Institute
|
| Department |
Medical Oncology
|
| Lab |
Shivdasani
|
| Street address |
44 Binney St.
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL339 |
| Series (1) |
| GSE6593 |
Expression analysis of primary mouse megakaryocyte differentiation |
|
