As described above, bioinformatics predictions for switchgrass miRNAs might be limited by the lack of large genomic data and the number of available ESTs. Direct cloning is a possible experimental approach to discover not only conserved, but also novel switchgrass-specific miRNAs. Several groups have used this method to identify plant miRNAs in different plant species (Sunkar and Zhu 2004; Sunkar et al. 2005; Yao et al. 2007; Sunkar et al. 2008; Zhao et al. 2010; Kulcheski et al. 2011; Li et al. 2012).

The cloning methods involve in small RNA library construction (including isolation of small RNAs, ligation of adaptor oligonucleotides, reverse transcription, and amplification) and sequencing. Matts et al.

(2010) pooled equal molar amounts of total RNA from three-month-old switchgrass seedlings and inflorescences for small RNA library construction. The amplification products were then subjected to pyrosequencing following Sunkar et al. (Sunkar et al. 2008). A total of 21,999 raw sequences was generated and subjected to further analysis to discard duplicates, degradation products from ribosomal RNAs, transfer RNAs, small nuclear RNAs and mRNAs (Sunkar et al. 2008; Matts et al. 2010). The remaining small RNAs were subsequently analyzed for distinguishing miRNAs from siRNAs, and conserved miRNAs were identified by searching against miRBase (Matts et al. 2010).