|
| Status |
Public on Nov 06, 2007 |
| Title |
Rpl7a_knockout_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Rpl7a deletion cells grown in rich media at 30C
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
RPL7A::KAN in BY4741 background
|
| Growth protocol |
50mL cultures were grown in YPD to early log phase (OD260 0.2-0.4) at 30oC., centrifuged at room temperature and flash-frozen using liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
mRNA was extracted from cell pellets using the hot phenol method and precipitated overnight in ethanol
|
| 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.
|
| |
|
| Hybridization protocol |
The purified cRNA is fragmented in order to facilitate the subsequent hybridization step. 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 |
Replicate #2 of rpl7a- mRNA sample used for analysis of ribosomal protein knockout expression levels.
|
| Data processing |
Gene Chip Operating System's Statistical Expression algorithm is used to analyze the cell intensities and generate both expression values and presence/absence calls
|
| |
|
| Submission date |
Aug 13, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Suzanne Komili |
| Organization name |
Harvard University
|
| Street address |
44 Binney St.
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL90 |
| Series (1) |
| GSE8761 |
Transcriptional profiling of ribosomal protein knockouts |
|
