[
East Asia Worm Meeting,
2004]
We isolated novel small RNAs from C. elegans by using gel electrophoresis techniques. Sequences of the separated RNAs were determined by cloning and sequencing their cDNAs. RNAs of about 50 to 1,000 nt in length were prepared from the mixed stage worms and separated by the denaturing gel electrophoresis. 32 bands were detected and sequences of 107 cDNAs from the bands were determined. Eighty-seven cDNAs corresponded to parts of the known ncRNA gene sequences such as rRNAs, tRNAs and U snRNAs. Nine cDNAs had parts of exon sequences and eight had parts of intron sequences of protein coding genes. The remaining 3 cDNAs revealed sequences corresponded to the intergenic sequences of genome. RNAs smaller than 50 nt in length were also separated on the denaturing gel and fifteen bands were detected. Although the purified RNAs from the 15 bands were subjected to the enzymatic sequencing method of Donis-Keller, clear sequence could not be obtained. This is probably because each band contains more than one RNA species, since more than 100 species of tiny RNAs (19-23 nt) are detectable by Northern blotting (Ambros et al., 2003 and Lim et al., 2003). This small RNA fraction was further separated by a two dimensional (2D) gel electrophoresis, which resolved about 100 spots. By using this 2D-gel electrophoresis, we compared the expression pattern of embryonic small RNAs with small RNAs prepared from the mixed stage worms. Remarkable difference between the two was observed. The spots were classified into three groups, 1) spots which are detected only in the embryonic RNA preparation, 2) spots which are detected only in the mixed stage worm RNA preparation, 3) spots which are detected both in the embyonic and mixed stage worm RNA preparations. Each group had 85, 51 and 54 spots, respectively. Several cDNA seqeuences, which contained novel small non-coding RNA candidates, were obtained from each group.