|
| Status |
Public on Mar 30, 2020 |
| Title |
5169_D3 |
| Sample type |
RNA |
| |
|
| Source name |
Whole blood
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Whole blood
sample_number: 5169
sample_type: Patient
diagnosis: septic shock
sample_day: D3
sample_batch: 13
|
| Treatment protocol |
Peripheral whole blood from ICU patients or healthy volunteers was collected from ICU patients or healthy volunteers was collected in PAXgeneTM Blood RNA tubes (PreAnalytix). Samples were stabilized at least 4h at room temperature after collection and frozen at -80°C following the manufacturer’s guidelines. For ICU patients, blood was collected at D1, D3 and D6, after ICU admission.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from whole blood using PAXgene Blood RNA kit (PreAnalytix) according to the manufacturer’s instructions. No sample with RNA integrity number ≤ 6 (bad RNA quality) was present.
|
| Label |
biotin
|
| Label protocol |
The cDNA synthesis and amplification steps were performed from 16 ng of RNA using the Ovation Pico WTA System V2 kit (Nugen). Briefly, cDNA synthesis was done using a mixture of random and polydT primers, followed by the synthesis of the complementary strand. The Single Primer Isothermal Amplification (SPIA) was then performed with hybrid DNA/RNA primers sensitive to RNAse-H digestion, in the presence of a DNA polymerase with strong strand displacement activity. The resulting amplified cDNA was purified using the QIAquick purification kit (Qiagen), from which, total DNA concentration was measured using the NanoDrop 1000 spectrophotometer (Thermo Scientific) and the product quality was checked on the Bioanalyser 2100 (Agilent).
|
| |
|
| Hybridization protocol |
5 micrograms of purified DNA were fragmented into 50-200 bp fragments and were 3-labeled using Encore Biotin Module kit (Nugen). The resulting target was mixed with standard hybridization controls and B2 oligonucleotides following recommendations of manufacturer. The hybridization cocktail was heat-denatured at 99°C for 2 minutes, incubated at 50°C for 5 minutes and centrifuged at 16,000 g for 5 minutes to pellet the residual salts. The HERV-V3 microarrays were prehybridized with 200 µL of hybridization buffer and placed under stirring (60 rpm) in an oven at 50°C for 10 minutes. The hybridization buffer was then replaced by the denatured hybridization cocktail. Hybridization was performed at 50°C for 18 hours in the oven under constant stirring (60 rpm). Washing and staining were carried out according to the protocol supplied by the manufacturer, using a fluidic station: GeneChip fluidic station 450 (Affymetrix).
|
| Scan protocol |
The arrays were finally scanned using a fluorometric scanner: GeneChip scanner 3000 7G (Affymetrix)
|
| Description |
RNA from whole blood
|
| Data processing |
CEL files were transformed into matrix, normalized, adjusted for background noise (RMA normalization) and probes were summarized into probesets with command apt-probeset-summarize (V 1.18.0) with rma option . Batch effects were removed using COMBAT. Probesets were filtered when log2 signal was under 5.5 in more than 68% of samples.
|
| |
|
| Submission date |
Oct 16, 2018 |
| Last update date |
Mar 30, 2020 |
| Contact name |
Julien Textoris |
| E-mail(s) |
[email protected]
|
| Phone |
+33 472 119 546
|
| Organization name |
bioMérieux
|
| Department |
Medical Diagnostic Discovery Department (MD3)
|
| Lab |
Joint Research Unit - bioMérieux / HCL
|
| Street address |
Hôpital Edouard herriot - Pavillon P; 5 place d'Arsonval
|
| City |
Lyon |
| ZIP/Postal code |
69437 |
| Country |
France |
| |
|
| Platform ID |
GPL22462 |
| Series (2) |
| GSE121350 |
Modulation of LTR-retrotransposons expression in septic shock patients: a pilot study [MIP] |
| GSE121352 |
Modulation of LTR-retrotransposons expression in mHLA-DR stratified septic shock patients: a pilot study |
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