RNA preparation, quantitative RT-PCR of miRNA/mRNA targets, miRNA array, and whole-transcriptome microarray

Total RNA was isolated from NRVM using Trizol Reagent (Invitrogen) according to the manufacturer’s instructions. RNA was also collected from human control and heart failure tissue samples. Tissue samples were first pulverized using liquid N₂ and mortar and pestle to assist in the homogenization with Trizol. Subsequent RNA was used as a template for cDNA synthesis in reverse transcriptase reactions using the Multiscribe Reverse Transcriptase kit (Invitrogen) for detecting both mRNAs and miRNAs. The quantitative PCR reactions were performed with the ABI 7500 Real-Time PCR system using either SYBR green technology for coding genes or Taqman reagent for detecting mature miRNAs. mRNAs were normalized with GAPDH or ribosomal protein 27 (RPL27) and miRNAs with small nucleolar RNA U87 or U6. All miRNA primer sets were designed and provided by Invitrogen Taqman Assays. Real-time PCR reactions were conducted using TaqMan Universal Master Mix II (Invitrogen). miRNA primer sets for real-time PCR detection were as follows:

Rat miR-34b: Assay name, mmu-miR-34b-5p; Assay ID, 002617; Catalogue #, 4427975

Human/Rat miR-34c: Assay name, hsa-miR-34c; Assay ID, 000428; Catalogue #, 4427975

Rat U87 (housekeeping gene): Assay name, U87; Assay ID, 001712; Catalogue #, 4427975

Human miR-34b: Assay name, hsa-miR-34b; Assay ID, 000427; Catalogue #, 4427975

Human U6 (housekeeping gene): Assay name, U6 snRNA; Assay ID, 001973; Catalogue #, 4427975

Primer sets used in the detection of mRNA transcripts were designed in Primer 3 Plus and specificity to the intended target verified using Primer Blast (NCBI).

Rat Scn5a

Forward primer, 5’-TCAATGACCCAGCCAATTACCT-3’, Reverse primer, 5’-CCCGGCATCAGAGCTGTT-3’

Rat Scn1b

Forward primer, 5’-ACGTGCTCATTGTGGTGTTAACC-3’, Reverse primer, 5’-CCGTGGCAGCAGCAATC-3’

Rat Kcnd3

Forward primer, 5’-GCCTTCGAGAACCCACA-3’, Reverse primer, 5’-GATCACCGAGACCGCAATG-3’

Rat Kcnip2

Forward primer, 5’-ACTTTGTGGCTGGTTTGTCG-3’, Reverse primer, 5’-TGATACAGCCGTCCTTGTTGAG-3’

Rat GAPDH

Forward primer, 5’-AGTTCAACGGCACAGTCAAG-3’, Reverse primer, 5’-ACTCCACGACATACTCAGCAC-3’

Rat Rpl27

Forward primer, 5’-GCTGTCGAAATGGGCAAGTT-3’, Reverse primer, 5’-GTCGGAGGTGCCATCATCAA-3’

Human Kcnip2

Forward primer, 5’-TGTACCGGGGCTTCAAGAAC-3’, Reverse primer, 5’-GGCATTGAAGAGAAAAGTGGCA-3’

Human Scn5a

Forward primer, 5’- CTGCGCCACTACTACTTCACCAACA-3’, Reverse primer, 5’- TCATGAGGGCAAAGAGCAGCGT-3’

Human Scn1b

Forward primer, 5’- GACCAACGCTGAGACCTTCA-3’, Reverse primer, 5’- TCCAGCTGCAACACCTCATT-3’

Human Kcnd3

Forward primer, 5’- TCAGCACGATCCACATCCAG-3’, Reverse primer, 5’- CTCAGTCCGTCGTCTGCTTT-3

Human GAPDH

Forward primer, 5’- TCCTCTGACTTCAACAGCGA-3’, Reverse primer, 5’- GGGTCTTACTCCTTGGAGGC-3’.

RNA collected from NRVM following KChIP2 and control siRNA treatment were submitted to miRNA microarray analysis to determine miRNAs regulated by the loss of KChIP2. The array was performed by the Gene Expression and Genotyping Facility at Case Western Reserve Univesity using the Affymetrix GeneChip miRNA 4.0 array. The resulting. CEL files were used with ExpressionConsole to conduct RMA analysis to derive the relative intensities of the miRNA probe set. The raw datasets are available from the Gene Expression Omnibus (Accession {"type":"entrez-geo","attrs":{"text":"GSE75806","term_id":"75806"}}GSE75806).

Additionally, RNA collected from NRVM following KChIP2 silencing with an adeno-shRNA expression system with non-targeting (control) and KChIP2 targeting constructs were used to assess global gene changes following KChIP2 loss. A total of 1.5 x 10⁶ cells were plated on 35 mm dishes. Cells were cultured in DMEM/5% FBS/penicillin/sptreptomycin with 0.1 mM BrdU for 24 hrs. After 24 hrs, media was replaced with fresh DMEM/5% FBS/penicillin/sptreptomycin and the corresponding control and KChIP2 shRNA virus. Cells were cultured for 48 hrs (with a media change after 24 hrs) and collected for total RNA and evaluated using a whole-transcriptome microarray. The array was performed by the Gene Expression and Genotyping Facility at Case Western Reserve University using the Affymetrix rat Clariom S Assay. The resulting. CEL files were used with Expression Console and the Transcriptome Analysis Console provided by Affymetrix (available here: http://www.affymetrix.com/support/technical/software_downloads.affx) to derive the relative changes in gene expression. The raw datasets are available from the Gene Expression Omnibus (Accession {"type":"entrez-geo","attrs":{"text":"GSE94623","term_id":"94623"}}GSE94623)