Wang et al. BMC Genomics 2011, 12:289 http://www.biomedcentral.com/1471-2164/12/289 Page 7 of 14 Figure 5 Fold change of unique sequences and the abundance of Chinese cabbage csRNAs in the HT seedlings. Validation of csRNAs in chloroplasts To investigate whether the csRNAs are located in the chloroplast, we isolated the highly purified chloroplasts of Chinese cabbage and Arabidopsis and performed North- ern blotting. For comparison, the RNA samples from the intact and broken chloroplasts were separated. The lack of nuclear RNA contamination was confirmed by PCR after reverse transcription, using primers designed for (i) At4g10760 (the nuclear gene encoding the chloroplast MTA protein) and (ii) At4g15030 (encoding the nuclear TCP4 protein) gene sequences (Figure 8A). csRNA-4.5sr-1 (sequence in Additional File 9) was the most abundant csRNA family, and its csRNAs were derived from the 3’ end of the 4.5S rRNA in chloroplasts with its 3’ termini located -1 to +1 nt relative to the last nucleotide of the rRNA. In the csRNA datasets of Chinese cabbage, three abundance peaks at ~18, 22 and 25 nt were displayed con- sistently for the HT and MT seedlings (Figure 8B). Using Northern blotting analyses, the 18 nt csRNAs and the other csRNAs belonging to csRNA-4.5sr-1 were identified in the intact and broken chloroplast RNAs. Nevertheless, the csRNA-4.5sr-1 csRNAs in the broken chloroplast were less abundant than in the intact chloroplasts (Figure 8C). Similarly, the csR-23sr-1 csRNAs in the broken chloro- plasts of Arabidopsis were less abundant than in the intact chloroplasts (Figure 8E). In addition, amount of ~16 nt csR-23sr-1 csRNAs is in agreement with our Arabidopsis deep sequencing results (Figure 8D). We noticed several RNA molecules of 80-200 nt in size that might be the multiple precursors (Figure 8C and 8E). Apart from the observations above, one small RNA core sequence, csRNA-ig-1, was represented by a large number of reads at an average size of 22 nt in each of the Chinese cabbage csRNA datasets and a specific 24 nt size in the Arabidopsis chloroplast dataset, which Figure 6 Identification of csR-23sr-1 and csR-5sr-1. (A) and (C) Size distribution analysis of the csR-23sr-1 family and the csR-5sr-1 family in sequencedatasets with core sequences (underlined in Additional File 9). (B) and (D) Northern blotting analysis of csR-23sr-1 and csR-5sr-1 in RNA samples from heat-treated and control seedlings of Chinese cabbage. U6 spliceosomal RNA was used as an RNA loading control. mapped to one intergenic RNA locus but nowhere else. This small RNA sequence is identical to the Ntc-2 small non-coding RNA reported and verified by Lung and col- leagues in their tobacco chloroplast study [10]. Interest- ingly, csRNA-ig-1 was induced under heat stress (Additional Files 8 and 9).