|
| Status |
Public on May 15, 2017 |
| Title |
Lean 4 Subcutaneous |
| Sample type |
RNA |
| |
|
| Source name |
Lean 4 Subcutaneous Adipose
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Adipose
disease state: Lean
bmi: 22
age: 13
race: Mexican
Sex: Female
|
| Treatment protocol |
No Treatment
|
| Growth protocol |
Human obese and lean visceral and subcutaneous fat were surgically acquired, dissected, and promptly cultured for 60 minutes.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
To isolate exosomes, we used the cell culture supernatant from the adipose tissue cultures and used ExoQuick-TC precipitation solution, according to ExoQuick-TC protocol. Total RNA was isolated following mirVana's total RNA Isolation kit.
|
| Label |
biotin
|
| Label protocol |
We used Affymetrix FlashTag Biotin HSR RNA Labeling Kit to label 1000 ng of cRNA from each sample.
|
| |
|
| Hybridization protocol |
For hybridization, we followed Affymetrix Gene Chip miRNA Array Procedure for Affymetrix miRNA 3.0 arrays to hybridize biotin-labeled sample.
|
| Scan protocol |
We scanned the Affymetrix miRNA 3.0 arrays following the Affymetrix Command Console User Manual Instructions.
|
| Description |
Lean subcutaneous fat was promplty cultured after surgery for 60 minutes. Cell culture supernatant was used to isolate exosomes.
|
| Data processing |
We analyzed the resulting data in Expression Console using RMA+DMBG (Affymetrix) and then exported it to Partek Genomics Suite for analyses. We used three-factor ANCOVA (ID*group (lean vs. obese)*adipose depot; age covariate) to compare miRNA expression, using unadjusted p ≤ 0.05 and fold change ≥ |1.2| as filters. We generated main effects for group (obesity-state) and tissue depot along with their interaction term. As a part of the overall ANCOVA, we used specific within-group (depot effects within obese or lean) and within-tissue (obesity effects within depot) contrasts. No corrections were made for multiple testing, as the primary purpose of list generation was to power pathway analysis, which mitigates false positive discovery risks.
|
| |
|
| Submission date |
Sep 04, 2013 |
| Last update date |
May 15, 2017 |
| Contact name |
Robert Freishtat |
| E-mail(s) |
rfreishtat@childrensnational.org
|
| Organization name |
Children's National Medical Center
|
| Department |
Center for Genetic Medicine
|
| Street address |
111 Michigan Avenue NW
|
| City |
Washington |
| State/province |
DC |
| ZIP/Postal code |
20010 |
| Country |
USA |
| |
|
| Platform ID |
GPL16384 |
| Series (1) |
| GSE68885 |
Adipocyte-derived Exosomal miRNAs from Subcutaneous Adipose Tissue: A Novel Mechanism for Obesity-Related Disease |
|
