|
| Status |
Public on Jun 30, 2017 |
| Title |
SCD_HbSS_CS_2 |
| Sample type |
RNA |
| |
|
| Source name |
Peripheral venous blood
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Whole blood
gender: Male
age: 14y
state: crisis
|
| Treatment protocol |
Peripheral venous blood was collected from each subject through phlebotomy, in a falcon tube containing 0.05 mg of Ethylenediaminetetraacetic acid (EDTA), under aseptic conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from whole blood by using QIAamp® RNA Blood Mini Kit (Qiagen). The target of RNA isolation from the whole blood was leukocytes which include lymphocytes, monocytes and granulocytes. The RBCs were not targeted for the same as they do not contain nuclei, and RNA is not synthesized in them. Only the RNA species larger than 200 nucleotides were isolated and rest other small RNAs were discarded as they do not bind to the QIAamp spin column. RNA sample was quantified by electrophoresing in Agilent 2100 Bio-analyzer, version G2938C on Agilent RNA 6000 Nano Kit (Agilent Technologies). Only, the RNA samples having RIN values ≥7 were subjected to the next step of the experiment.
|
| Label |
Cy3
|
| Label protocol |
cDNA was synthesized from 600 ng of total RNA using Agilent one color RNA spike-In kit and Agilent Quick Amp Kit, One-Color. cRNA synthesis and cyanine 3 (Cy3) labeling was done using the same. Labeled cRNA samples were subjected to purification using Qiagen RNeasy® Mini Kit (Qiagen). Purified labeled cRNA samples were quantified on NanoDrop ND-1000 UV-VIS Spectrophotometer version 3.2.1 and Agilent 2100 Bio-analyzer
|
| |
|
| Hybridization protocol |
Cy3 labeled cRNA was hybridized using Gene Expression Hybridization Kit (Agilent Technologies). The labeled cRNA samples (targets) were hybridized to the fixed probes on the Whole Human Genome (4×44K) Oligo Microarray kit (Human GE 4×44K V2 Microarray kit), using Agilent Hybridization chamber and backing slides. Microarray slides were incubated at 65°C for 17 hours in a hybridization oven. Microarray slides were washed using Gene Expression Wash Buffers 1 and 2, to discard partially hybridized and unhybridized samples. A surfactant Triton X-102(10%) was added to buffers 1 and 2 to remove background artifacts from microarray slide without affecting the stringency of wash buffers.
|
| Scan protocol |
The microarray slides were scanned using a DNA Microarray Scanner with Surescan High- Resolution Technology (Agilent Technologies) equipped with extended dynamic range (XDR) software.
|
| Description |
Gene expression of patients in crisis state
|
| Data processing |
Feature Extraction Software (Version 9.5.3) was used for data extraction from raw microarray image files (.tif). It was followed by feature extraction using Feature Extraction Software (Version 9.5.3). Data visualization, pre-processing, analysis and mining were performed using GeneSpring GX (Silicon Genetics, Redwood City, CA). During normaliztion raw signals were thresholded to 1.0; normalization algorithm used was scale; percentile target was chosen as 75. Scaling was done median of percentile target of all samples. Median value was chosen as preprocessing baseline for all samples.
|
| |
|
| Submission date |
Sep 14, 2015 |
| Last update date |
Jun 30, 2017 |
| Contact name |
Ipsita Das |
| E-mail(s) |
pkp1964@yahoo.co.in
|
| Organization name |
Pt. Jawaharlal Nehru memorial Medical College
|
| Department |
Biochemistry
|
| Lab |
Genetics
|
| Street address |
Jail Road
|
| City |
Raipur |
| State/province |
Chhattisgarh |
| ZIP/Postal code |
492001 |
| Country |
India |
| |
|
| Platform ID |
GPL13497 |
| Series (1) |
| GSE72999 |
Gene expression analysis of sickle cell disease patients |
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