|
| Status |
Public on Jan 23, 2024 |
| Title |
Human blood, CONTROL 29 |
| Sample type |
genomic |
| |
|
| Source name |
COVID-19 negative subjects_buffy coat
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: CONTROL
tissue: Blood
age (y): 73
Sex: female
|
| Treatment protocol |
DNA was extracted from buffy coat specimens collected within 48 hours after patient admission at the hospital. Briefly, EDTA blood samples were centrifuged at 2,500 x g at 4°C for 15 minutes, the buffy coat layer was removed and stored at -80°C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total DNA was extracted using a QIAamp DNA blood mini kit (Qiagen) following the manufacturer’s instructions.
|
| Label |
biotin
|
| Label protocol |
Total DNA was amplified by means of REPLI-g mitochondrial DNA kit (Qiagen) that contains DNA polymerase, buffers, and reagents for the specific amplification of the mitochondrial genome using Multiple Displacement Amplification (MDA). After purification, DNA was quantified spectrophotometrically and Genechip Resequencing array kit (Affymetrix) was employed for fragmentation and labeling.
|
| |
|
| Hybridization protocol |
The chips were hybridized for 16 h at 49°C, then washed and stained in Fluidics Station 450 according to the suggested protocol.
|
| Scan protocol |
Chips were scanned in Affymetrix GeneChip Scanner 3000 7G and data acquisition was performed using the Affymetrix Genechip Command Console (AGCC) software.
|
| Description |
Selective amplification of mtDNA
|
| Data processing |
Data analysis was carried out with GSEQ 4.1 with “model type” set at diploid to enable the detection of heteroplasmy and “quality score threshold” set at 3 to provide the best base calling accuracy and rate. The other parameters used by the algorithm were No Signal Threshold= 2, Weak Signal Fold Threshold= 20, Large SNR Threshold= 20, Min Fraction of Calls of Samples = 0.5, Trace Threshold = 1, Sequence Profile Threshold = -0.175.
Homoplasmic and heteroplasmic variants were defined by the software through comparison with the revised Cambridge Reference Sequence (rCRS). Probe Intensity File values were used to calculate the Ratio of Expected Allele (REA).
|
| |
|
| Submission date |
Jun 22, 2023 |
| Last update date |
Jan 23, 2024 |
| Contact name |
Tiziana Casoli |
| E-mail(s) |
t.casoli@inrca.it
|
| Phone |
+39 071 8004203
|
| Organization name |
INRCA
|
| Department |
Scientific Technological Area
|
| Lab |
Center for Neurobiology of Aging
|
| Street address |
Via Birarelli 8
|
| City |
Ancona |
| ZIP/Postal code |
60121 |
| Country |
Italy |
| |
|
| Platform ID |
GPL10983 |
| Series (1) |
| GSE235626 |
Association of inflammatory mediators with mitochondrial DNA mutations in geriatric COVID-19 patients |
|
