Targeted myocardial gene expression in failing hearts by RNA sequencing.

BACKGROUND: Myocardial recovery with left ventricular assist device (LVAD) therapy is highly variable and difficult to predict. Next generation ribonucleic acid (RNA) sequencing is an innovative, rapid, and quantitative approach to gene expression profiling in small amounts of tissue. Our primary goal was to identify baseline transcriptional profiles in non-ischemic cardiomyopathies that predict myocardial recovery in response to LVAD therapy. We also sought to verify transcriptional differences between failing and non-failing human hearts. METHODS: RNA was isolated from failing (n = 16) and non-failing (n = 8) human hearts. RNA from each patient was reverse transcribed and quantitatively sequenced on the personal genome machine (PGM) sequencer (Ion torrent) for 95 heart failure candidate genes. Coverage analysis as well as mapping the reads and alignment was done using the Ion Torrent Browser Suite™. Differential expression analyses were conducted by empirical analysis of digital gene expression data in R (edgeR) to identify differential expressed genes between failing and non-failing groups, and between responder and non-responder groups respectively. Targeted cardiac gene messenger RNA (mRNA) expression was analyzed in proportion to the total number of reads. Gene expression profiles from the PGM sequencer were validated by performing RNA sequencing (RNAseq) with the Illumina Hiseq2500 sequencing system. RESULTS: The failing sample population was 75% male with an average age of 50 and a left ventricular ejection fraction (LVEF) of 16%. Myosin light chain kinase (MYLK) and interleukin (IL)-6 genes expression were significantly higher in LVAD responders compared to non-responders. Thirty-six cardiac genes were expressed differentially between failing and non-failing hearts (23 decreased, 13 elevated). MYLK, Beta-1 adrenergic receptor (ADRB1) and myosin heavy chain (MYH)-6 expression were among those significantly decreased in failing hearts compared to non-failing hearts. Natriuretic peptide B (NPPB) and IL-6 were significantly elevated. Targeted gene expression profiles obtained from the Ion torrent PGM sequencer were consistent with those obtained from Illumina HiSeq2500 sequencing system. CONCLUSIONS: Heart failure is associated with a network of transcriptional changes involving contractile proteins, metabolism, adrenergic receptors, protein phosphorylation, and signaling factors. Myocardial MYLK and IL-6 expression are positively correlated with ejection fraction (EF) response to LVAD placement. Targeted RNA sequencing of myocardial gene expression can be utilized to predict responders to LVAD therapy and to better characterize transcriptional changes in human heart failure.

Major alteration in coxsackievirus B3 genomic RNA structure distinguishes a virulent strain from an avirulent strain.

Coxsackievirus B3 (CV-B3) is a cardiovirulent enterovirus that utilizes a 5' untranslated region (5'UTR) to complete critical viral processes. Here, we directly compared the structure of a 5'UTR from a virulent strain with that of a naturally occurring avirulent strain. Using chemical probing analysis, we identified a structural difference between the two 5'UTRs in the highly substituted stem-loop II region (SLII). For the remainder of the 5'UTR, we observed conserved structure. Comparative sequence analysis of 170 closely related enteroviruses revealed that the SLII region lacks conservation. To investigate independent folding and function, two chimeric CV-B3 strains were created by exchanging nucleotides 104-184 and repeating the 5'UTR structural analysis. Neither the parent SLII nor the remaining domains of the background 5'UTR were structurally altered by the exchange, supporting an independent mechanism of folding and function. We show that the attenuated 5'UTR lacks structure in the SLII cardiovirulence determinant.

A formalin-free method for stabilizing cells for nucleic acid amplification, hybridization and next-generation sequencing.

BACKGROUND: Formalin has been widely used by pathology laboratories. Its carcinogenicity has led researchers to explore formalin substitutes. Streck Cell Preservative (SCP) is a formalin-free preservative that can preserve cellular antigens. This study was undertaken to investigate the effects of cell preservation using SCP on nucleic acid amplification, hybridization, and next-generation sequencing (NGS) as compared to control frozen cells and cells fixed in the traditional cell and tissue fixative, 10 % neutral buffered formalin (NBF). FINDINGS: The breast cancer cell line, SKBR-3, was used as a model system. Prior to nucleic acid extraction and fluorescence in situ hybridization (FISH), cells were fixed in SCP or NBF overnight at room temperature with frozen cells in parallel. Analysis showed that similar DNA extraction yields and amplification profiles determined by PCR in SCP preserved cells and control frozen cells, whereas NBF preserved cells had decreased DNA yield and impaired PCR amplification. Molecular cytogenetic studies by FISH technique indicated that the ratios of ERBB2 (HER-2/neu) signals to the chromosome 17 centromere (CEP17) were comparable for frozen cells and SCP preserved cells. The fluorescence images of both SCP fixed and control frozen cells were also clear and comparable. On the contrary, the same analysis was unsuccessful with NBF preserved cells due to poor hybridization quality. Our data also demonstrated that SCP had negligible effect on NGS testing. CONCLUSION: We conclude that SCP can be used as an alternative to NBF as a preservative for maintaining the integrity of nucleic acids for nucleic acid amplification, sequencing and FISH analysis.