|
| Status |
Public on Oct 15, 2017 |
| Title |
C05 Post |
| Sample type |
RNA |
| |
|
| Source name |
C05 Post Serum
|
| Organism |
Homo sapiens |
| Characteristics |
biological fluid: Serum
bmi pre: 40
bmi post: 20.7
age: 49
race: AF
Sex: Female
|
| Treatment protocol |
No Treatment
|
| Growth protocol |
Human bariatric patient pre and post surgery serum (500 uL) was used for isolation of FABP4+ exosomes.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
To isolate exosomes, we used the serum from the bariatric patients (pre and post surgery) and used ExoQuick-TC precipitation solution, according to ExoQuick-TC protocol. Total RNA was isolated following mirVana's total RNA Isolation kit. We also used StemCell Technologies Magnetic Isolation Technique to isolate FABP4+ specific exosomes.
|
| 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 4.0 arrays to hybridize biotin-labeled sample.
|
| Scan protocol |
We scanned the Affymetrix miRNA 4.0 arrays following the Affymetrix Command Console User Manual Instructions.
|
| Description |
FABP4+ exosomes (adipocyte-derived) were isolated, amplified, and profiled from serum taken pre bariatric surgery.
|
| 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 |
Oct 15, 2015 |
| Last update date |
Oct 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 |
GPL19117 |
| Series (1) |
| GSE74083 |
Circulating Adipose-derived Exosomal miRNAs and metabolites associated with insulin signaling are altered by gastric bypass surgery |
|
