Wang et al. BMC Genomics 2011, 12:289 http://www.biomedcentral.com/1471-2164/12/289 Page 6 of 14 Figure 4 position-of-origin analysis of Chinese cabbage csRNA. To normalize the varying rRNAs or tRNAs length, RNA sequences were divided into 5% blocks from the 5’ to the 3’ ends. The flanking sequence outer of each ends were calculated proportionally. The positions of csRNA 3’ ends and 5’ ends were plotted into rRNA (A) and tRNA (B) blocks, respectively. (C) The peak of 3’ ends of rRNA-derived csRNAs at the 3’ termini of rRNA. (D) The peak of 5’ ends of tRNA-derived csRNAs at the 5’ termini of tRNA. from 23S and 5S rRNAs, respectively, (sequences in Additional File 9). In Chinese cabbage, csR-23sr-1 are predominantly 26 and 21 nt, while csR-5sr-1 are typi- cally 26 nt in length, and they were reduced after heat treatment (Figure 6A and 6C). To test how heat stress affects accumulation of csR-5sr-1 and csR-23sr-1 csRNAs, we performed Northern hybridization of small RNAs. In the HT seedlings, 26 nt csR-23sr-1 and 26 nt csR-5sr-1 csRNAs accumulated much less than in the MT seedlings (Figure 6B and 6D). These data suggest that accumulation of rRNA-derived csRNAs is affected by heat stress. As a whole, the tRNA-derived csRNAs only slightly declined in the HT seedlings compared to those in the MT seedlings (Figure 5). However, the length of these csRNAs was related to heat response (Additional File 4C and 4D). In the HT seedlings, the abundance of the longer csRNAs (29-32nt) decreased, but that of the shorter ones (16-25nt) increased compared to those in the MT seedlings (Table 2). For each tRNA, abundance of the short csRNAs increased while that of the longer csRNAs declined except for tRNA-Gly. csR-trnA-1 and csR-trnA-2 are two most predominant tRNA-derived csRNA families, and originated from chloroplast tRNA- Ala (Figure 7A). The former showed two peaks (17 and 29 nt) while the latter displayed only one peak (23 nt) (Figure 7B and 7C) (sequences in Additional File 9). Northern blotting showed that abundance of 29 nt csR- trnA-1 csRNAs under heat stress declined while those of 17 nt csRNAs increased (Figure 7D), and abundance of 23 nt csR-trnA-2 csRNAs remarkably increased (Fig- ure 7E), consistent with the deep sequencing result. Like rRNA-derived csRNAs, tRNA-derived csRNAs are affected by heat stress. In addition to the above-mentioned origins, small RNAs of 21~24 nt from mRNAs and igRNAs increased in HT seedlings compared to those in the control lines (Additional File 4E-H), and those showing relatively high abundances were verified by a microRNA microar- ray analysis (Additional File 9).