|
| Status |
Public on Jan 19, 2022 |
| Title |
Brain_AD_sample3 |
| Sample type |
RNA |
| |
|
| Source name |
brain tissue
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: AD
age: 79
gender: m
tissue: brain
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated by Trizol™ method (Invitrogen, Karlsruhe, Germany). 100mg deeply frozen human brain tissue (temporal cortex) was homogenized in the presence of 1ml Trizol in a glass-Teflon™ homogenizer. The homogenate was transferred to a microtube and after adding chloroform, samples were centrifuged at 15 000 g (4 °C) for 15min and the supernatant transferred to a fresh tube. Samples were mixed with equal amounts of isopropanol and centrifuged at 12 000 g (4 °C) for 15min to precipitate the RNA. After washing, the pellet was air-dried and dissolved in water. RNA quality was assessed by denaturing formaldehyde agarose gel electrophoresis, by spectrophotometry (scanning at 220−320 nm) and by analysis using Agilent 2100 bioanalyzer. Only samples with RIN > 5 were further processed. The RNA concentration was estimated spectrophotometrically by absorbance at 260 nm, concentration was adjusted to 1mg/ml and RNA was stored at −80 °C until use.
|
| Label |
Cy3
|
| Label protocol |
1 μg of total RNA was labeled using the Quick Amp Labeling Kit (Agilent, Waldbronn, Germany), according to the manufacturer’s instructions with the adaptation of using 120 pmol of a random N6 − T7 primer (Metabion, Planegg, Germany) instead of a polyT-T7 primer. cRNA quantity was checked using a NanoDrop ND-1000 UV-VIS Spectrophotometer, as enlisted in the manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
1.65 μg of labeled cRNA was used for hybridization following manufacturer’s instructions.
|
| Scan protocol |
After hybridization the arrays were washed according to the manual and scanned using the Agilent G2565CA Microarray Scanner System with Agilent Scan Control Software (Version A851) following settings for scanning: Profile: AgilentG3 GX 1Color; Channels Green; Scan Region: Agilent HD (61 × 21.6mm); Resolution 3 μm double pass; Tiff: 20 bit; Green PMT Gain: 100%. Result tables were extracted after grid placement using Agilent Feature Extraction Software (Version 11.5.1.1)
|
| Data processing |
Differential expression analysis was performed using R and the Bioconductor package Limma. Quality control of arrays was performed by checking distribution of “bright corner”, “dark corner” probes, and relative spike-in concentration versus normalized signal. The controls confirmed high quality of the results and consequently all microarray data were included in the downstream analysis. Initially, independent filtering was performed, removing probes (i) with signal intensity above the background in less than one third of all arrays and (ii) exhibiting an interquartile range of log2 signal intensity across all samples of less than 1. Background expression was defined by the mean intensity plus three times the standard deviation of negative control spots (Agilent’s 3xSLv spots). 113 047 out of 931 898 probes were retained after filtering. Signal intensities were quantile normalized but not background corrected, due to the low background intensities of Agilent arrays. Differential expression between AD and control samples was determined using a linear model that includes age because on average, individuals from the AD group were older than controls. The linear model was fitted using the R package limma and reliable variance estimates were obtained by Empirical Bayes moderated t-statistics. False discovery rate was controlled by a modified Benjamini-Hochberg procedure that incorporates an estimated proportion of the null p-values 23 to compute q-values using the fdrtool R package.
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| |
|
| Submission date |
Oct 01, 2019 |
| Last update date |
Jan 19, 2022 |
| Contact name |
Kristin Reiche |
| E-mail(s) |
kristin.reiche@izi.fraunhofer.de
|
| Organization name |
Fraunhofer Institute for Cell Therapy and Immunology
|
| Department |
Diagnostics
|
| Street address |
Perlickstr. 1
|
| City |
Leipzig |
| ZIP/Postal code |
04103 |
| Country |
Germany |
| |
|
| Platform ID |
GPL27556 |
| Series (2) |
| GSE138260 |
Differential expression analysis of human brain samples with Alzheimer disease versus healthy control samples |
| GSE138261 |
Alzheimer-related genes show accelerated evolution |
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