|
| Status |
Public on Jul 31, 2012 |
| Title |
hTERT BPE Biological Replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
hTERT_BPE_Biological Replicate 3
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: hTERT immortalized meibomian gland epithelial cells
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with Trizol reagent, followed by clean-up with QIAGEN RNeasy mini kit in accordance with the prescribed protocol provided the manufacturers. Quality control was performed with Agilent Bioanalyser.
|
| Label |
Biotin
|
| Label protocol |
Biotinylated cRNA were prepared using a MessageAmp™ II-based protocol (Ambion Inc., Austin, TX) and one round of amplification. The cRNA yields were quantified by UV spectrophotometry and the distribution of transcript sizes was assessed using the Agilent Bioanalyzer 2100 capillary electrophoresis system. Labeled cRNA was used to probe HumanHT-12 v3 Expression BeadChips.
|
| |
|
| Hybridization protocol |
cRNA/hybridization buffer mixtures were incubated at 65°C for 5 minutes, followed by brief centrifugation to collect. The mixtures were added to BeadChips and then BeadChips inserted into hybridization chambers, and hybridization was carried out at 58°C for 16-18 hours at rocking speed set to 5. BeadChips were washed and stained in the following steps: 1) 55°C for 10 minutes static in HighTemp Wash Buffer in a Hybex waterbath, 2) ambient temperature shaking for 5 minutes in E1BC wash buffer, 3) ambient temperature shaking for 10 minutes in ethanol, 4) ambient temperature shaking for 2 minutes in E1BC buffer, 5) ambient temperature for 10 minutes rocking in Block E1 buffer, 6) ambient temperature for 10 minutes rocking in Block E1 buffer containing 1ug/ml Cy3-streptavidin, and 7) ambient temperature shaking for 5 minutes in E1BC buffer.
|
| Scan protocol |
BeadArrays were dried in a centrifuge for 4 minutes and scanned using a BeadArray scanner. Illumina BeadScan software was used to produce .idat, .xml, and .tif files for each array on a slide and .sdf files for each barcode on a slide of 12 arrays. Raw data were extracted using Illumina BeadStudio software v 3. Following quality assessment, data from the replicate beads on each array were summarized into raw intensity values with and without background subtraction in an Excel report containing the project description (sample key), gene identifiers and corresponding probe IDs, table of detection p-values, tables of normalized data with and without background subtraction, array quality control metrics, and array quality control metric cut-offs and definitions.
|
| Description |
replicate 3
|
| Data processing |
Data were were cubic spline normalized using Illumina Bead Studio software . After normalization the data were furhter processed by changing all negative values to zero and then adding 16 to each intensity.
|
| |
|
| Submission date |
Sep 14, 2009 |
| Last update date |
Jul 31, 2012 |
| Contact name |
David A Sullivan |
| E-mail(s) |
david.sullivan@schepens.harvard.edu
|
| Phone |
(617) 912-0287
|
| Fax |
(617) 912-0101
|
| Organization name |
Schepens Eye Research Institute
|
| Lab |
David Sullivan
|
| Street address |
20 Staniford Street
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02114 |
| Country |
USA |
| |
|
| Platform ID |
GPL6947 |
| Series (1) |
| GSE18099 |
Growth factor influence on the gene expression profile of human meibomian gland epithelial cells |
|
