|
| Status |
Public on May 21, 2019 |
| Title |
Human blood, PD 1 |
| Sample type |
genomic |
| |
|
| Source name |
Parkinson's patient
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: PD
tissue: Whole blood
locomotion mode: 0
adl hierarchy scale: 1
|
| Treatment protocol |
The blood samples used in the study came from 15 PD patients (mean age 78.7 ± 6.1; 8 females), treated with levodopa, and 15 age- and sex-matched controls (mean age 80.9 ± 5.1; 9 females). All participants were of Caucasian ethnicity. The PD patients showed late disease onset (≥ 65 years) and no family history of PD. Exclusion criteria for PD patients were a history of other neurological disorders, a diagnosis of PD as a secondary disease, and PD with atypical disease progression. Exclusion criteria for control subjects were a history of any neurological disease, orthostatic hypotension, cognitive impairment, and parkinsonism.
Controls consisted in volunteers and spouses of the patients and were submitted to the same clinic and neuropsychological assessment as the PD group. Each participant underwent blood withdrawal with sodium citrate as anticoagulant.
|
| 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) and the heteroplasmy percentage.
|
| |
|
| Submission date |
Apr 26, 2018 |
| Last update date |
May 21, 2019 |
| 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) |
| GSE113704 |
Mitochondrial DNA allelic substitutions in Parkinson’s disease |
|
