Agilent rna 6000 nano assay

Total RNA was extracted from ventricular myocardium using the TRI Reagent (Molecular Research Center Inc., Cincinnati, OH) as described previously (11 (link)). The concentration of RNA was determined using Agilent RNA 6000 Nano assay (Agilent technologies Inc., Germany) following the manufacturer’s instruction. For RT reaction, 1 μg of RNA was used as a template to generate complementary DNA using the SuperScript III First-Strand Synthesis Kit (Invitrogen), and the RT was performed by following the manufacturer’s instructions. For the duplex PCR of a gene of interest and GAPDH, 2 μl of solution from the RT reaction and specific primers toward the target gene and GAPDH were used. The mRNA levels of the gene of interest were quantified by PCR at the minimum number of cycles (15 cycles) capable of detecting the PCR products within the linear amplification range. Mouse PDE1A, GFPdgn, and GAPDH were measured using the following primer pairs: PDE1A, 5′-CTAAAGATGAACTGGAGGGATCTTCG GAAC-3′ (forward) and 5′-TGGAGAAAATGGAAGCCCTAATTCAGC-3′ (reverse); GFPdgn, 5′-TCTATATCATGGCCGACAAGCAGA-3′ (forward) and 5′-ACTGGGTGCTCAGGTAGTGGTTGT-3′ (reverse); and GAPDH, 5′-ATGACATCAAGAAGGTGGTG-3′ (forward) and 5′-CATACCAGGAAATGAGCTTG-3′ (reverse).

For microarray analysis, we included two mice from each group to achieve biological replicates. Total RNA was extracted from the LV using TRIzol (Sigma-Aldrich), and RNA integrity was analyzed using the Agilent RNA 6000 Nano assay. Qubit was used to determine RNA quality. The same amount of total RNA (100 ng) was used for each sample. Targets were prepared using an Eberwine-based amplification method with the One Array plus RNA amplification kit (Phalanx Biotech Group, CA San Diego, USA) to generate amino-allyl antisense RNA. Aminoallyl-RNA probes labeled with NGS-Cy5 were hybridized at 50°C for 16 h to the Mice Whole Genome One Array Version 2.0. The hybridized array was scanned with the Agilent Microarray Scanner (G2505 C) under 100% and 10% PMT, respectively, at a 10 μM resolution. After scanning, the raw intensity data were analyzed with Gene Pix™ 4 to determine mRNA levels. The data were pre-processed using the Rosetta Resolver® System (Rosetta Biosoftware). Differentially expressed genes were those with a log₂ (fold change) ≥1 and P < .05.