|
| Status |
Public on Dec 26, 2019 |
| Title |
KP160 |
| Sample type |
RNA |
| |
|
| Source name |
Medium Arsenic exposed subjects (Mean Urinary As. 84.77±9.36 µg/g.creat)
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Whole Blood
gender: Male
age group: 26-39 years
smoking status: Non smokers
residence type: Rural
|
| Treatment protocol |
Freshly taken blood samples were taken RNA later and within two hours of samples collection, samples were stored at -80C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from the samples taken in RNA later was isolated using Ambion Ribopure-Blood RNA Kit (Cat#AM1928) as per manufacturer guidelines. Extracted RNA was quantified using Nanodrop-1000 Spectrophotomer and RNA quality was checked through Agilent 2100 Bioanalyzer
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labelled cRNA was prepared from 200ng Total RNA using Agilent one colour Quick Amp Labeling kit according to manufacturer protocol followed by Qiagen RNAeasy Column Purification. Purified cRNA yeild was measured through Nanodrop-1000 Spectrophotometer
|
| |
|
| Hybridization protocol |
600ng of Cy3 Labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25µl containing 1X Agilent Fragmentation Buffer, 10X Blocking Agent followed by 25µl of 2X Hi-RPM Hybridization buffer to stop the fragmentation reaction as per manufacturer guidelines for Agilent Sureprint G3 Human GE Kit 8 x 60K V2 G4851B Microarrays. Hybridization Assembly wase made as per guidelines of Agilent microarrays Hybridization Chamber User guide (G2534-90001). 40µl of Hybridized sample was loaded on slide in Chamber and Incubated for 17 hours in Hybridization oven at 65°C. Gene expression wash buffers containg 10% Triton-X102 were used for washing. Microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation
|
| Scan protocol |
Immediately after washing, slides were scanned on the Agilent DNA Microarray Scanner using one color scan setting for 8x60k array slides. Pixel intensities were extracted as raw data from the scan images using Agilent Feature Extraction Software (Agilent) using default parameters
|
| Description |
Peripheral Blood Stored in RNA later
Gene expression analysis of rural subjects exposed to elevated Arsenic levels in drinking water
|
| Data processing |
All arrays were checked for their quality using ArrayQC (https://github.com/BiGCAT-UM/arrayQC_Module/), a quality control pipeline in R (version 2.10.1; The R Foundation for Statistical Computing, Vienna, Austria). For each spot the following steps were taken: local background correction, flagging of bad spots, controls and spots with too low intensity, log2 transformation, and quantile normalization. We filtered out control probes and low expressed probes. To get an idea of how bright expression probes should be, we computed the 95% percentile of the negative control probes on each array. We kept probes that are at least 10% brighter than the negative controls on at least 18 arrays (i.e. smallest number of subjects per exposure group). Next the avereps function was used to average replicate spots.
|
| |
|
| Submission date |
Feb 20, 2018 |
| Last update date |
Dec 26, 2019 |
| Contact name |
Muhammad Yasir Abdur Rehman |
| E-mail(s) |
mahryasir@gmail.com
|
| Phone |
+923515357447
|
| Organization name |
Quaid-i-Azam University
|
| Department |
Environmental Sciences
|
| Lab |
Environmental Health Laboratory
|
| Street address |
1
|
| City |
Islamabad |
| State/province |
ICT |
| ZIP/Postal code |
45320 |
| Country |
Pakistan |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE110852 |
Gene expression profiles of subjects exposed to Arsenic through drinking water in Pakistan |
|
