Abstract
BackgroundRNA extraction is a crucial step for monitoring gene expression. Poor RNA quality (including degradation and remaining impurities) can result in misleading results. Isolation of RNA from animal tissues with high lipid content can be challenging. Especially, it is not trivial to isolate high quality RNA with a reasonable yield from adipose tissue. The aim of this study was to provide an optimized protocol for isolating total RNA from adipose tissue. This was achieved by combining the advantages of the two routinely used methods, TRI Reagent® and miRNeasy.FindingsThe miRNeasy method results in cleaner samples but more prone to degradation while the TRI Reagent® method results in samples contaminated with salts and solvents but more intact. The new protocol combines the best of both methods resulting in RNA of high quality and suitable for downstream experiments like RT-qPCR, microarrays and high-throughput sequencing.ConclusionsThe current protocol for total RNA isolation from adipose tissue yields sufficient amount of high quality total RNA free of contaminants.
Highlights
RNA extraction is a crucial step for monitoring gene expression
Due to its high content in fatty acids and in some instances its low cell number, adipose tissue poses some challenges when high quality RNA needs to be isolated for downstream gene expression applications
Verification of the RNA integrity by microfluidics (Experion or Bioanalyzer) is crucial in order to evaluate if the isolated RNA is of sufficient quality for downstream applications [9]
Summary
RNA extraction is a crucial step for monitoring gene expression. Poor RNA quality (including degradation and remaining impurities) can result in misleading results. The aim of this study was to provide an optimized protocol for isolating total RNA from adipose tissue This was achieved by combining the advantages of the two routinely used methods, TRI ReagentW and miRNeasy. Due to its high content in fatty acids and in some instances its low cell number, adipose tissue poses some challenges when high quality RNA needs to be isolated for downstream gene expression applications (i.e. arrays, high-throughput sequencing or RT-qPCR). Many routinely used protocols for isolation of RNA from adipose tissue result in RNA that is partially degraded and/or poor yield of RNA or the small RNAs are lost in the process All these issues can cause misleading results and they are a big challenge specially when working with
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