strain: C57BL/6 tissue: Sinoatrial node tissue: Pool of 4 SAN age: 3 months
Treatment protocol
Experimental_protocol:Animal studies were performed in accordance with the 1986 British Home Office Animals Scientific Procedures Act (UK) incorporating European Directive 2010/63/EU, the European Directive (86/609/CEE) on the care and use of laboratory animals, and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication no. 85-23, revised 1996). All experimental protocols were assessed and approved by the local university ethical review committee. Sinoatrial node (SAN) isolation. The heart with lungs was quickly removed and immersed at 4 °C to wash out the blood in an external solution that contained (in mmol/L): NaCl, 137; KCl, 4.9; NaH2PO4, 1.2; glucose,15; HEPES, 20; MgCl2, 1.0; pH 7.4. The heart-lung block was pinned to the tissue bath to excise the right atrium (RA) and SAN under a stereomicroscope. The tissue bath was perfused with the external solution at a rate of 10 mL/min. After removal of both ventricles and the left atrium, the RA was opened to expose the crista terminalis, the intercaval area and the interatrial septum. This preparation was pinned by small stainless steel pins to the chamber with the endocardial side exposed up and trimmed carefully to extract the SAN region correctly. The SAN region was delimited by the borders of the crista terminalis, the interatrial septum, superior and inferior vena cava. All tissues were snap-frozen in liquid nitrogen and stored at -80 °C for subsequent RNA extraction. AMPK R299Q γ2 knock-in mice were created through gene-targeting to introduce the R299Q point mutation (equivalent to the human R302Q mutation) into exon 7 of murine Prkag2. Mice heterozygous for the R299Q γ2 knock-in mutation were initially on a mixed C57BL/6/129/Ola genetic background and subsequently backcrossed to C57BL/6 for at least 7 generations. Four mouse SAN tissues were pooled from R299Q γ2 and WT littermate controls and processed together for the extraction of one sample of total RNA for a total of 4 homozygote R299Q γ2, 4 heterozygote R299Q γ2 and 3 WT samples.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted with an RNeasy Mini Kit (Qiagen) using DNase on-column digestion according to the manufacturer’s protocol, and assessed for quality and quantity using an Agilent 2100 Bioanlyzer with RNA 6000 Nano Chips, (Aligent technologies).
Label
biotin-Cy3
Label protocol
Standard Illumina protocol using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) In short, 0.5ug of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.
Hybridization protocol
Standard Illumina protocol. In short, a total of 0.75ug of biotin-labeled cRNA was hybridized at 58 degrees C for 16 hours to Illumina's Mouse-Ref8 v2 Expression Bead Chips (Illumina, San Diego, CA). Each BeadChip has ~25,600 well-annotated RefSeq transcripts . The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3.
Scan protocol
Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina.
Description
SAN_Het2
Data processing
Data was extracted using the Illumina GenomeStudio software V2011.1, Gene expression module 1.9.0. Any spots at or below the background were filtered out using an Illumina detection Pvalue of 0.02 and above. Data was analyzed using two different normalization approaches. For z-score normalized data, the natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated. Z-score = (raw value - avg)/std. For Robust Spline Normalisation, all data were log2-transformed and normalised using the lumi software package in Bioconductor. 18,185 nucleotide probes were filtered from 25,697 total using an Illumina detection P value of α = 0.05. Significant genes were selected by a one-way ANOVA or t-test (FDR = 0.05) for further analysis. GSEA software was used to select differentially expressed (DE) genes by calculating out a ‘score’ using the Kolmogorov-Smirnov (KS) test. The score indicates how a gene relates with genotype, where positive values relate to up-regulation and negative values relate to down-regulation. Only transcripts with a KS score > |0.15| were considered DE. Transcripts demonstrating greater than 1.2-fold change in expression were processed using Ingenuity Pathway Analysis software (Ingenuity Systems, CA, USA) to identify networks and canonical pathways overrepresented in enriched genes.Complete data including detection scores is supplied.
