RNA extraction from healthy and failing human myocardium: a comparative evaluation.

BACKGROUND: Isolation of high-quality RNA from tissue is mandatory for producing reliable data for downstream applications. In heart tissue, the relative strengths and weaknesses of different approaches to isolate total RNA are unknown. The objective of this study was to compare different RNA isolation methods in healthy and diseased human myocardium. METHODS: Frozen left ventricular myocardium was obtained from individuals with heart failure and individuals who died from non-cardiac causes with normal heart function (control). Three extraction methods, including guanidine isothiocyanate (TRIzol), silica-gel column (RNeasy), and the combination method (TRIzol/RNeasy), were assessed for their effect on the yield, integrity, and gene expression levels of RNA using quantitative real-time PCR. RESULTS: In the control group (n=5), the highest RNA yield per tissue mass was obtained with TRIzol, and a significantly higher RNA integrity was obtained from the RNeasy method. The quantification cycle (Cq) values for both the reference gene GAPDH and two target genes were lower with TRIzol. Normalization by GAPDH showed the highest gene expression levels with RNeasy. Similar patterns were observed in the heart failure group (n=5), suggesting assays were not negatively impacted by myocardial disease processes. CONCLUSION: In both healthy and diseased heart tissue, the TRIzol method provides the highest RNA yield, while the RNeasy method shows superior RNA integrity, demonstrating comparable RNA quality in studies examining myocardial disease. A balanced approach to RNA quality is necessary for the successful downstream applications of RNA.

Increased expression of FoxM1 transcription factor in respiratory epithelium inhibits lung sacculation and causes Clara cell hyperplasia.

Foxm1 is a member of the Forkhead Box (Fox) family of transcription factors. Foxm1 (previously called Foxm1b, HFH-11B, Trident, Win, or MPP2) is expressed in multiple cell types and plays important roles in cellular proliferation, differentiation and tumorigenesis. Genetic deletion of Foxm1 from mouse respiratory epithelium during initial stages of lung development inhibits lung maturation and causes respiratory failure after birth. However, the role of Foxm1 during postnatal lung morphogenesis remains unknown. In the present study, Foxm1 expression was detected in epithelial cells of conducting and peripheral airways and changing dynamically with lung maturation. To discern the biological role of Foxm1 in the prenatal and postnatal lung, a novel transgenic mouse line that expresses a constitutively active form of FoxM1 (FoxM1 N-terminal deletion mutant or FoxM1-ΔN) under the control of lung epithelial-specific SPC promoter was produced. Expression of the FoxM1-ΔN transgene during embryogenesis caused epithelial hyperplasia, inhibited lung sacculation and expression of the type II epithelial marker, pro-SPC. Expression of FoxM1-ΔN mutant during the postnatal period did not influence alveologenesis but caused focal airway hyperplasia and increased proliferation of Clara cells. Likewise, expression of FoxM1-ΔN mutant in conducting airways with Scgb1a1 promoter was sufficient to induce Clara cell hyperplasia. Furthermore, FoxM1-ΔN cooperated with activated K-Ras to induce lung tumor growth in vivo. Increased activity of Foxm1 altered lung sacculation, induced proliferation in the respiratory epithelium and accelerated lung tumor growth, indicating that precise regulation of Foxm1 is critical for normal lung morphogenesis and development of lung cancer.

Chondroitin sulfate proteoglycans are required for lung growth and morphogenesis in vitro.

Proteoglycans (PGs) have been shown to play a key role in the development of many tissues. We have investigated the role of sulfated PGs in early rat lung development by treating cultured tissues with 30 mM sodium chlorate, a global inhibitor of PG sulfation. Chlorate treatment disrupted growth and branching of embryonic day 13 lung explants. Isolated lung epithelium (LgE) migrated toward and invaded lung mesenchyme (LgM), and chlorate irreversibly suppressed this response. Chlorate also inhibited migration of LgE toward beads soaked in FGF10. Chlorate severely decreased branching morphogenesis in tissue recombinants consisting of LgM plus either LgE or tracheal epithelium (TrE) and decreased expression of surfactant protein C gene (SP-C). Chlorate also reduced bone morphogenetic protein-4 expression in cultured tips and recombinants but had no effect on the expression of clara cell 10-kDa protein (CC10), sonic hedgehog (Shh), FGF10, and FGF receptor 2IIIb. Chlorate reduced the growth of LgE in mesenchyme-free culture but did not affect SP-C expression. In contrast, chlorate inhibited both rudiment growth and the induction of SP-C in mesenchyme-free cultured TrE. Treatment of lung tips and tissue recombinants with chondroitinase ABC abolished branching morphogenesis. Chondroitinase also suppressed growth of TrE in mesenchyme-free culture. Chondroitinase treatment, however, had no effect on the induction of SP-C expression in any of these cultures. These results demonstrate the overall importance of sulfated PGs to normal lung development and demonstrate a dynamic role for chondroitin sulfate PGs in embryonic lung growth and morphogenesis.