MicroRNAs have emerged as important post-transcriptional regulators of gene expression. Identi fi cation of cancer-regulated microRNAs or other classes of endogenous small RNAs have advanced our knowledge in cancer progression and metastasis. Among many tools, small RNA cloning is a powerful method to identify new microRNAs (miRNAs) and to pro fi le miRNA expression and function. Retroviral system is also the minimum requirement for the studying of miRNAs in a highly stable population of cancer cells or other primary cell types with high expression. This chapter describes a step-by-step protocol that is optimized to clone directly one of the miRNA miR-145, as an example, from genomic DNA into retroviral vector to yield ultimate overexpression for functional study in prostate cancer cells. The small RNAs cloned by this protocol will have an easy and simple way of cloning from genomic DNA to maintain the necessary motifs of native enhancer for enhancement of mature miRNA expression. Furthermore, the procedure eliminates miRNA extraction and cDNA synthesis before cloning and sequential cloning of more than one miRNA makes this protocol cost-and time-effective to eliminate many frequent cell culturing.