MicroRNA Profiling During Human Keratinocyte Differentiation Using a Quantitative Real-Time PCR Method

The terminal differentiation of epidermal keratinocytes requires transcriptional and posttranscriptional regulatory mechanisms. MicroRNAs (miRNAs) are small noncoding RNAs that play key roles during differentiation processes by regulating protein expression at the posttranscriptional level. Several studies have investigated miRNA expression in murine or human skin using northern blotting, microarrays, deep sequencing, or real-time PCR (Andl et al., Curr Biol 16:1041–1049, 2006; Hildebrand et al., J Invest Dermatol 131:20–29, 2011; Sonkoly et al., PLoS One 2:e610, 2007; Yi et al., Nat Genet 38:356–362, 2006; Yi et al., Proc Natl Acad Sci U S A 106:498–502, 2009). Conventional techniques such as northern blotting and microarrays often fail to detect miRNAs of low abundance, while the per-sample cost of deep sequencing approaches is still prohibitive in many cases. In contrast, stem loop primer-based real-time PCR methods for simultaneous detection of up to 380 miRNAs allow fast, specific, and reliable miRNA profiling. These methods are suitable for in vitro material, but also for samples which are of limited availability, such as epidermal stem cells isolated from human skin biopsies. Here, we describe a general real-time PCR method for miRNA profiling using isolated epidermal stem cells, transiently amplifying cells and terminally differentiated keratinocytes of human skin.