Premium
P3‐243: Gene expression profiling of postmortem brain tissues affected by Alzheimer's disease
Author(s) -
Miyashita Akinori,
Saito Yuko,
Kakita Akiyoshi,
Ogishima Soichi,
Tanaka Hiroshi,
Takahashi Hitoshi,
Murayama Shigeo,
Ihara Yasuo,
Kuwano Ryozo
Publication year - 2008
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2008.05.1810
Subject(s) - rna , trizol , gene expression , human brain , 18s ribosomal rna , microbiology and biotechnology , gene , microarray , rna extraction , biology , pathology , chemistry , medicine , biochemistry , neuroscience
Background: Most common form of Alzheimer’s disease (AD) is late-onset AD (LOAD) ( 60 y.o.), accounting for roughly 95% of the case. Molecular mechanism of LOAD is not well understood. A high dense DNA microarray is available to analyze gene expression profiles of frozen tissues. Here we characterize total RNA isolated from human postmortem brain tissues prior to setting out identifying novel genes contributing to development or progression of LOAD. Methods: Two independent institutes of Japan, Tokyo Metropolitan Institute of Gerontology (N 48) and Niigata University (N 77), collected human postmortem brain tissues and quickly frozen at -80 degrees Celsius until use. We isolated total RNA from 125 brain tissues using the Trizol reagent and then the crude RNA was purified with an on-column DNase I treatment by a spin column purification system (Invitrogen). Quantification of the purified RNA was fluorometrically conducted using the Quant-iT RiboGreen RNA reagent (Molecular Probes). RNA quality was assessed by capillary gel electrophoresis (2100 BioAnalyzer, Agilent). An RNA integrity number (RIN, the best score is 10) and the ratio of 28S/18S ribosomal RNA were calculated with Expert software (Agilent). Results: Eighty-four of the 125 brain tissues showed 7.0 of the RIN. The RIN (mean [SD], 7.3 [1.2]; range, 3.4 9.3) was strongly correlated with the 28S/18S ratio (mean [SD], 1.0 [0.3]; range, 0.3 1.7) (r-square 0.6852, P 0.0001). Both age at death (mean [SD], 79.7 y.o. [10.6]; range, 44 98 y.o.) and postmortem interval (mean [SD], 7.2 hr [9.9]; range, 1.0 64.1 hr) did not show any significant correlation between the RIN and the 28S/18S ratio. Conclusions: We have assessed total RNA isolated from postmortem human brain tissues, and observed that the RNA is high quality suitable for gene expression analysis. Gene expression profiling analysis is in progress using a DNA microarray, where over 47000 transcripts and variants spanning the entire human genome can be surveyed. It is expected that novel transcripts and its related proteins responsible for LOAD could be found through the expression profiling using the total RNA characterized here.