Abstract
As microRNAs (miRs) are gaining increasing attention as key regulators of cellular processes, expressional quantification is widely applied. However, in the processing of relatively quantified data, the importance of testing the stability of several reference mRNAs and/or miRs and choosing among these for normalization is often overlooked, potentially leading to biased results. Here, we have optimized the purification of miR-enriched total RNA from pancreatic insulin-producing INS-1 cells. Additionally, we optimized and analyzed miR expression by a qPCR-based microarray and by specific qPCR and tested the stability of candidate reference mRNAs and miRs. Hence, this study gives a widely applicable example on how to easily and systematically test and decide how to normalize miR quantification. We suggest that caution in the interpretation of miR quantification studies that do not comprise stability analysis should be exerted.
Highlights
Characterizing the expressional levels of microRNA in various tissues and cell lines, and under different conditions, is increasingly performed by microarrays and quantitative PCR on purified miR [1]
We use miR quantification from the insulin-producing cell line INS-1 to illustrate the importance of systematic optimization of RNA purification and quantitation of miRs from a specific sample type, including profiling and selection of suitable reference candidates
We took advantage of INS-1 cells stably transfected with lentivirally transduced short hairpin RNA knockdown of histone deacetylases HDAC1, HDAC2, HDAC3, or empty vector (EV) shown previously to affect responses to inflammatory and metabolic stress [14,15], since knockdown of these key transcriptional regulators were expected to modify miR expression
Summary
Characterizing the expressional levels of microRNA (miR) in various tissues and cell lines, and under different conditions, is increasingly performed by microarrays and quantitative PCR (qPCR) on purified miR [1]. The choice of one or more housekeeping genes or miRs for normalization of qPCR data is crucial to avoid potential technical bias. We use miR quantification from the insulin-producing cell line INS-1 to illustrate the importance of systematic optimization of RNA purification and quantitation of miRs from a specific sample type, including profiling and selection of suitable reference candidates. In several studies on miRs in pancreatic β cells, traditionally used housekeeping genes, such as U6 and RNU6B, have been applied as endogenous controls for normalization, notably without any given justification for the choice of reference [9,10,11,12]. The present study is to our knowledge the first published systematic optimization of miR quantification in pancreatic β cells
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