Differential expression of small RNAs in soybean

Small RNAs are non-protein coding RNAs that regulate gene expression in plants primarily by cleaving mRNA. Two major classes of small non-coding RNAs are microRNAs (miRNAs) and short interfering RNAs (siRNAs) of 21-24 nucleotides. I selected some small RNAs that show tissue specificity based on results from high-throughput sequencing for further investigation. I optimized the protocols for small RNA isolation and blotting. Purified RNAs from multiple tissues such as immature seed coats and cotyledons, germinated cotyledons, unifoliates, shoot tips, stems, and roots were used to examine differential expression. The RNA blots were probed with the 5’ end-labeled oligonucleotides that would complement the sequence of the small RNAs. One dramatic example is represented by the endogenous tissue-specific siRNAs that are present only in the immature seed coats of soybean varieties with yellow seed coats (Tuteja et al., 2009) and that target chalcone synthase mRNAs for down regulation leading to absence of pigment in the seed coats. In addition, several other novel miRNAs and siRNAs were shown to be expressed differentially in seed coats versus cotyledon including highly abundant small RNAs with over 100,000 sequences per million total sequence reads