RNA integrity and quality.

<p>RNA quality indicator (RQI) for hippocampal slices and percentage of samples for each RQI value range.</p

Relative expression of the main receptor and channel genes involved in excitatory and inhibitory hippocampal neurotransmission systems during A<i>β</i> incubation.

<p>Expression levels of mRNA for <b>(A)</b> glutamatergic <b>(B</b>) cholinergic and <b>(C)</b> GABAergic genes, at 0, 30 and 120 min after ACSF (control) or A<i>β</i> incubation, were analyzed by qPCR and normalized to <i>Ppia</i>, the most stably expressed reference gene in our study. Normalized 2^–ΔCq expression for each gene and time point is presented as percentage of gene expression at time 0 (t = 0 min) which is considered as 100% basal control expression. <b>(A)</b>. Relative mRNA expression levels of glutamatergic NMDA receptor subunits <i>GluN1</i>, <i>GluN2A</i> and <i>GluN2B</i>, AMPA receptor subunits <i>GluA1</i> and <i>GluA2</i>, and metabotropic receptor <i>mGlu1</i> and <i>mGlu5</i>. <b>(B)</b> Relative mRNA expression levels of GABA_B receptor subunits <i>GABA</i>_<i>B1</i> and <i>GABA</i>_<i>B2</i>, and GABA_B receptor effector GIRK channel subunits <i>Girk1</i>, <i>Girk2</i>, <i>Girk3</i> and <i>Girk4</i>. <b>(C)</b> Relative mRNA expression levels of cholinergic M1 receptor and M1 receptor effector KCNQ channel subunits <i>Kcnq2</i>, <i>Kcnq3</i> and <i>Kcnq5</i>. <b>(D)</b> Relative mRNA expression levels of activity-regulated cytoskeleton-associated protein, <i>Arc</i>, at 0, 30 and 120 min after ACSF or A<i>β</i> incubation are also shown. Note that y-scale in D is different to A-C. Data are presented as mean ± standard error of the mean (SEM). *<i>p</i> < 0.05. A<i>β</i>, amyloid-beta; <i>Ppia</i>, Peptidylprolyl isomerase A; <i>ACSF</i>, artificial cerebrospinal fluid.</p

Experimental design, RNA integrity category examples and reference gene selection.

<p><b>(A)</b> Horizontal hippocampal brain slices from one hemisphere were obtained using a dissecting microscope. Thick dashed lines indicate the cutting delimitation area. C, Caudal; L, Lateral. <b>(B)</b> Schematic representation of hippocampal slices incubation procedure. Slices were incubated in ACSF or A<i>β</i> 1°μM during 30 or 120 min. At time zero, hippocampal slices were considered as raw tissue and mRNA level for these samples 100% since incubation effects had no begun. All experiments were performed at room temperature and bubbled with carbogen gas. <b>(C)</b> Representative examples of electropherogram of different RNA samples revealing RNA integrity levels and virtual images of the gels for each sample. Left column (M), molecular weight marker; Right column (S), RNA sample. RQI values are ranged from 10 (intact) to 1 (totally degraded). The gradual degradation of RNA extracted from hippocampal slices was reflected by a continuous shift towards shorter fragment sizes. The first peak found in all traces corresponds to a molecular weight marker. Electropherogram plots from top to bottom: (Top) Profile of RNA with RQI = 9.1. The peaks correspond to 18S and 28S ribosomal subunits. In this case, there are no small peaks in profile which would indicate RNA degradation. Two bands corresponding to the 28S and 18S ribosomal RNA respectively. The greater thickness of the band corresponding to 28S indicates higher concentration compared to 18S subunit. (Middle) Profile of RNA with RQI = 7.1. Arrows indicate the different peaks from degraded RNA fragments which appear at different time points along the reaction. In the sample are arrowed degraded fragments of different sizes. (Bottom) RNA profile with RQI = 2.1. Plot corresponding to highly degraded RNA can be observed. No bands can be distinguished. <b>(D)</b> The BestKeeper and NormFinder softwares were used to calculate the most stable gene among the 3 reference genes, in ACSF and A<i>β</i> incubation. For both softwares the most stable gene is that with the lowest stability value. Asterisk indicates the selected gene (Ppia) as the most stably expressed reference gene. <i>Actb</i>, β-actin; <i>Gapdh</i>, Glyceraldehyde 3-phosphate dehydrogenase, <i>Ppia</i>, Peptidylprolyl isomerase A.</p