|
| Status |
Public on Mar 11, 2014 |
| Title |
Human blood, Control 8 |
| Sample type |
genomic |
| |
|
| Source name |
blood, Age-matched control
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: Age-matched control
tissue: Whole blood
iadl: 8
mmse: 29
|
| Treatment protocol |
Subjects were enrolled from October 2009 to September 2010 at the Geriatrics Operative Unit of INRCA in Fermo (Italy). Patients were diagnosed according to NINCDS-ADRDA criteria for probable AD by means of an extended neuropsychological and functional evaluation, neuroimaging and laboratory tests.
Controls consisted in volunteers and spouses of the patients and were submitted to the same clinic and neuropsychological assessment as the AD 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 |
Sample name: C427
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.
Output files utilized for this study were the SNP View and Probe Intensity Files that provide, for each np, the base calls classified as homoplasmic or heteroplasmic and values of fluorescence intensity of the four bases for both sense and antisense filaments. The quantitative estimates of allelic contribution were used to calculate REA (Ratio of Expected Allele) index which is the log ratio of the signal intensity of the reference allele at any site, as indicated in the RCRS, to the average signal intensity of the other three alleles from the sense and antisense strand.
|
| |
|
| Submission date |
Jul 24, 2013 |
| Last update date |
Mar 11, 2014 |
| 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) |
| GSE49160 |
Increased contribution of non-reference alleles in mitochondrial DNA (mtDNA) of Alzheimer's disease (AD) patients |
|
