Journal: Frontiers in Molecular Neuroscience
Article Title: Region-Specific Differences in Amyloid Precursor Protein Expression in the Mouse Hippocampus
Figure Lengend Snippet: Quantitative App mRNA expression in hippocampal subregions. (A,B) LMD of hippocampal subregions, i.e., CA1 pcl and gcl of the DG. Representative section of the dorsal hippocampus (coronal plane, cresyl violet staining) before (A) and after (B) LMD is shown. Scale bar: 250 μm. (C) RNA integrity analysis of total RNA isolated from the dissected gcl (red) and from pcl (blue) demonstrating highly intact RNA (RIN-values: ~9; Agilent 2100 Bioanalyzer). (D,E) Average expression stability values ( M ) and evaluation of the optimum number of candidate reference genes for CA1 pcl and for DG gcl according to geNorm software. Pairwise variation (V) of candidate reference genes indicates that the use of the two most stable genes is sufficient to obtain an accurate normalization index for quantitative PCR (qPCR) analysis. (F) A significantly higher gene expression for App (1.5- to 1.7-fold) was detected in CA1 pcl relative to DG gcl using two different App -specific TaqMan assays ( A : Mm_01344172_m1, B : Mm_00431830_m1) after normalization to a reference gene index calculated by geNorm. (G,H) Gene expression stability values ( S ) and accumulated SD analysis using NormFinder. The minimal number of reference genes required for effective normalization is highlighted. (I) Comparable to the results obtained by geNorm algorithm, a significantly higher App expression (1.6- to 1.7-fold) was detected in CA1 pcl relative to DG gcl after normalization to the reference gene index estimated by NormFinder. Data ( N = 5–6 mice) were tested for statistical significance using one-way ANOVA followed by Bonferroni post hoc test to correct for multiple comparisons, * p ≤ 0.05.
Article Snippet: PCR products were checked on Agilent DNA 1000 Chips (Agilent Technologies) with the Agilent 2100 Bioanalyzer system to verify product specificity and amplicon size.
Techniques: Expressing, Laser Capture Microdissection, Staining, Isolation, Software, Real-time Polymerase Chain Reaction, Mouse Assay