RESUMEN
The accurate quantification of cellular and tissue mRNA and microRNA content is reliant upon the selection of stable endogenous control transcripts for normalizing quantitative real-time-PCR (qRT-PCR) data. Using the combination of unbiased and informed approaches and a wide range of human adipose tissues and cells, we sought to identify invariant control transcripts for mRNA and microRNA. A total of 26 mRNA transcript candidates were selected from the literature. MicroRNA candidates were selected from a microRNA-microarray (Agilent, n = 22 tissues), and together with candidates from the literature resulted in 14 different microRNAs. The variability of these mRNA and microRNA transcripts were then tested in a large (n = 180) collection of a variety of human adipose tissues and cell samples. Phosphoglycerate kinase-1 (PGK1) and peptidylprolyl isomerase A (PPIA) were identified as the most stable mRNAs across all tissues and panels. MiR-103 was overall the most stable microRNA transcript across all biological backgrounds. Several proposed and commonly used normalization transcripts were found to be highly variable. We then tested the effect on expression of two established adipocyte-related transcripts (fatty acid binding protein 4 (FABP4) and microRNA-145 (miR-145)), either normalized to the optimal or a commonly used controls transcript. This test clearly indicated that spurious results could arise from using less stable control transcripts for mRNA and microRNA qRT-PCR.
Asunto(s)
Tejido Adiposo/citología , MicroARNs/análisis , ARN Mensajero/análisis , Adipocitos/química , Células Cultivadas , Proteínas de Unión a Ácidos Grasos/genética , Perfilación de la Expresión Génica , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Análisis por Micromatrices , Isomerasa de Peptidilprolil/genética , Fosfoglicerato Quinasa/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: The metabolic defects underlying familial combined hyperlipidaemia (FCHL) are not clearly understood. We used stable isotope techniques combined with tissue-specific measurements in adipose tissue and forearm muscle to investigate fatty acid handling by these tissues in the fasting and postprandial states. RESULTS: Patients were insulin resistant as shown by higher glucose and insulin concentrations and lower muscle glucose extraction than controls. Plasma triacylglycerol (TAG) concentrations were higher in patients. Adipose tissue TAG extraction was not lower in patients than controls, although TAG clearance was lower, probably representing saturation. Following a test meal, patients showed a greater increase in chylomicron-TAG concentrations. There were no differences between FCHL patients and controls in postprandial suppression of non-esterified fatty acid (NEFA) concentrations or postprandial NEFA release, but patients had greater trapping of exogenous fatty acids in adipose tissue. 3-Hydroxybutyrate concentrations were lower in patients indicative of decreased hepatic fatty acid oxidation. CONCLUSIONS: In this group of patients with FCHL, the major defect appeared to be overproduction of TAG by the liver due to decreased fatty acid oxidation, with fatty acids directed to TAG synthesis. We found no evidence of decreased lipoprotein lipase action or impaired fatty acid re-esterification in adipose tissue.