Despite the growing interest in cardiac miRNA expression profiling, having high quality and yield in RNA extraction from cardiac tissue is still challenging. We compared different methods of tissue homogenization and total RNA extraction from pig cardiac tissue aimed at miRNAs expression profiling. Small biopsies of right atrial appendages were obtained from pig hearts and treated according to four different protocols: no homogenization (P1) and homogenization by manual (P2) or automatic (P3 and P4) methods, followed by Proteinase K digestion (PKD) except in P4. Total RNA was extracted using miRNeasy mini kit, assessing RNA yield and quality by Nanodrop. cDNA synthesis and qRT-PCR were performed using TaqMan MicroRNA Assay. Homogenization was crucial to obtain high yield of pure total RNA. Automatic methods displayed higher yield (0.27 μg RNA/mg tissue in P3) than manual (0.06 μg RNA/mg tissue in P2), with better performance without PKD step (0.38μg RNA/mg tissue in P4). RNA from P4 was suitable for miRNA expression profiling, as demonstrated by qRT-PCR on miRNA 21 and 29. These results suggest the efficacy of an automatic homogenization to extract RNA suitable for miRNA expression profiling.

Comparison of different methods to extract RNA from cardiac tissue for miRNA profiling by qRT-PCR

D'Amato, Elvira;Tessarolo, Francesco;Masè, M.;Ravelli, Flavia
2013

Abstract

Despite the growing interest in cardiac miRNA expression profiling, having high quality and yield in RNA extraction from cardiac tissue is still challenging. We compared different methods of tissue homogenization and total RNA extraction from pig cardiac tissue aimed at miRNAs expression profiling. Small biopsies of right atrial appendages were obtained from pig hearts and treated according to four different protocols: no homogenization (P1) and homogenization by manual (P2) or automatic (P3 and P4) methods, followed by Proteinase K digestion (PKD) except in P4. Total RNA was extracted using miRNeasy mini kit, assessing RNA yield and quality by Nanodrop. cDNA synthesis and qRT-PCR were performed using TaqMan MicroRNA Assay. Homogenization was crucial to obtain high yield of pure total RNA. Automatic methods displayed higher yield (0.27 μg RNA/mg tissue in P3) than manual (0.06 μg RNA/mg tissue in P2), with better performance without PKD step (0.38μg RNA/mg tissue in P4). RNA from P4 was suitable for miRNA expression profiling, as demonstrated by qRT-PCR on miRNA 21 and 29. These results suggest the efficacy of an automatic homogenization to extract RNA suitable for miRNA expression profiling.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11582/304116
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