|
| Status |
Public on Dec 01, 2020 |
| Title |
blood-6Month-ScoreA-patientCN220714 |
| Sample type |
RNA |
| |
|
| Source name |
venous blood-6Month-ScoreA-patientCN220714
|
| Organism |
Homo sapiens |
| Characteristics |
patient: CN220714
time: M6
MADRS: A
tissue: Venous blood
|
| Treatment protocol |
5ml venous blood were collected from the 57 patients, using vacuum tubes which included a reagent that immediately stabilized intracellular RNA (PAXgene blood RNA system, PreAnalytiX GmbH, Hombrechtikon, Switzerland). The samples were kept at room temperature for 8 h as required for stabilizing RNA and then at -20°C until RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with the PAXgene Blood RNA kit (Qiagen, Courtaboeuf, France) according to the manufacturer's protocol. Total RNA quality was assessed and its concentration was measured using RNA Nano chips onto a Bioanalyser 2100 (Agilent, Boeblingen, Germany).
|
| Label |
Cy3
|
| Label protocol |
For each sample, Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng of total RNA using the One-Color Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions, followed by Agencourt RNAClean XP (Agencourt Bioscience Corporation, Beverly, Massachusetts) purification. Dye incorporation and cRNA yield were checked using Dropsense™ 96 UV/VIS droplet reader (Trinean, Belgium).
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| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >6 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 10x Agilent fragmentation buffer and 25x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 Human GE v2 microarray (8X60K, Design 039494 enclosed in Agilent SureHyb-enabled hybridization chambers for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed sequentially in Wash buffer 1 (Agilent Technologies, 1 min) and Wash buffer 2 (Agilent Technologies, 37°C, 1 min).
|
| Scan protocol |
Slides were scanned immediately after washing on a Agilent G2505C Microarray Scanner with Agilent Scan Control A.8.5.1 software
|
| Description |
s44
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent) using default parameters (protocol GE1_1010_Sep10 and Grid: 039494_D_F_20120628). All subsequent data analyses were done under R (www.r-project.org) using packages of Bioconductor (www.bioconductor.org). Raw data (median of pixels intensity) were imported into R using the read.maimages function from the limma package with the following weight function (assigning a weight of 1 or 0 to each spot): myfunw<-function(x) {okType<-x$ControlType==0; okFoundGreen<-x$gIsFound==1; okPos=x$gIsPosAndSignif==1; okWellAbove<- x$gIsWellAboveBG==1; as.numeric(okType & okFoundGreen & okPos & okWellAbove);} We selected the spots with a minimal weight of 1 for 46 out of 57 microarrays or with a minimal weight of 6 per group (timePoint combined with MADRS score) from at least one experimental group. At this step, 33129 spots out of 62976 were selected. The mean signal of the 30 first samples and 27 last ones were substracted to individuals signals to correct the batch effect of the 2 serials observed during the microarray washing procedure. Data were then stored in an ExpressionSet object and normalized by the quantile method using the normalize.quantiles function from the preprocessCore R library. Replicated probes on the array (identical ProbeName) were resolved by taking the median normalized signal of each set of replicated probes. The resulting matrix has 27202 rows each corresponding to a unique ProbeName (provided as data Matrix).
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| |
|
| Submission date |
Jun 06, 2017 |
| Last update date |
Dec 01, 2020 |
| Contact name |
Aline FOURY |
| E-mail(s) |
aline.foury@inrae.fr
|
| Organization name |
INRAE
|
| Lab |
NutriNeuro
|
| Street address |
146 rue Léo Saignat
|
| City |
Bordeaux |
| ZIP/Postal code |
33600 |
| Country |
France |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE99725 |
Transcriptomic Signaling Pathways involved in a naturalistic model of Inflammation-related Major Depressive Disorder and its remission |
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