|
| Status |
Public on Mar 20, 2010 |
| Title |
beta-cells_non-diabetic condition_donor2 |
| Sample type |
RNA |
| |
|
| Source name |
human beta-cells_non-diabetic
|
| Organism |
Homo sapiens |
| Characteristics |
sample id: H16_2
disease: non-diabetic control
cell type: beta-cells
tissue: pancreas
gender: female
age (years): 65
bmi (body mass index, kg/m2): 34.0
|
| Treatment protocol |
Pancreas specimens were obtained, placed in cryomolds, embedded in Tissue-Tek OCT medium, immediately frozen in chilled isopentane, and stored at –80°C, pending sectioning at 8 μm. Frozen pancreatic sections were fixed in 70% ethanol for 30 seconds, rinsed by 5 dips in diethylpyrocarbonate (DEPC)-treated water and dehydrated in 100% ethanol twice for 1 min, and xylene for 4 minutes, the sections were air-dried and laser capture microdissection was performed. The microdissected cells were incubated with 10 μl of guanidine thiocyanate and polyethylene glycol octylphenol ether-based buffer (Buffer RLT, Qiagen), for 30 minutes at 42°C, and the samples stored at -80°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from each population of laser captured β–cells using phenol-chloroform-isoamyl alcohol and precipitated with sodium acetate and glycogen carrier in isopropanol. After initial recovery and resuspension of the RNA pellet, genomic DNA contamination was removed by incubation with 10 units of DNase I for 2 hours at 37°C, in presence of 10 units of RNase inhibitor. The treatment with DNase I was followed by RNA re-extraction and precipitation. The pellet was resuspended and total RNA was amplified by T7-based linear amplification using T7-oligo-dT-primers. Two rounds of amplification were performed using RiboAmp HS RNA Amplification Kits (Arcturus).
|
| Label |
biotin
|
| Label protocol |
Amplified RNA (1 μg) was converted into double-stranded complementary DNA (cDNA) using the RiboAmp HS RNA Amplification Kit (Arcturus), and biotinylated complementary RNA (cRNA) was generated from cDNA by in vitro transcription reaction using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics). RNA products were purified using the MiraColTM Purification Columns (Arcturus).
|
| |
|
| Hybridization protocol |
15ug of biotinylated cRNA was hybridized for 16hr at 45C to the Affymetrix Human X3P array. Following hybridization, arrays were washed and stained on the Affymetrix Fluidics Station 400.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.
|
| Description |
Gene expression data from control subjects
|
| Data processing |
Data were analyzed using DNA-chip Analyzer software (dChip), using the invariant set normalization algorithm.
|
| |
|
| Submission date |
Mar 19, 2010 |
| Last update date |
Mar 19, 2010 |
| Contact name |
Lorella Marselli |
| E-mail(s) |
Lorella.Marselli@med.unipi.it
|
| Phone |
+39 050 995135
|
| Fax |
+39 050 541521
|
| Organization name |
Harvard Medical School
|
| Department |
Joslin Diabetes Center and the Department of Medicine
|
| Lab |
Section on Islet Transplantation and Cell Biology
|
| Street address |
One Joslin Place
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL1352 |
| Series (1) |
| GSE20966 |
Gene expression profiles of beta-cell enriched tissue obtained by Laser Capture Microdissection from subjects with type 2 diabetes |
|
