Size selection and gel extraction of small RNAs from total RNA. Radioactively labeled markers and oligonucleotides are resolved with unlabeled total RNA on a 12% polyacrylamide gel; 24- and 28-nucleotide markers were run in a separate lane to allow for more accurate size resolution. Total RNA samples were spiked with a radiolabeled 24-nucleotide oligonucleotide (although any combination of labeled oligonucleotides can be used), allowing one to visually follow the ligation process and also ensuring that the desired size fraction has been extracted from the gel. Here, 20–28-nucleotide small RNAs (including the radiolabeled 24-nucleotide oligonucleotide) have been cut from the gel. The gel was then reexposed, showing the removal of radiolabeled oligonucleotides (and underlying small RNAs).