Wang et al. BMC Genomics 2011, 12:289 http://www.biomedcentral.com/1471-2164/12/289 Page 9 of 14 Figure 8 Validation of csRNAs in chloroplasts. (A) RT-PCR analysis of ATCG00020 (chloroplast gene encoding chloroplast psbA protein), At4g10760 (nuclear gene encoding chloroplast MTA protein) and At4g15030 (nuclear gene encoding the nuclear TCP4 protein). (B) and (C) Size distribution and Northern blotting of csR-4.5sr-1 in Chinese cabbage. (D) and (E) Size distribution and Northern blotting of csR-23sr-1 in Arabidopsis. Buffer: chloroplast isolation buffer mentioned in [47]; Br_IC: intact chloroplasts of Chinese cabbage. Br_BC: broken chloroplasts of Chinese cabbage; At_IC: intact chloroplasts of Arabidopsis; At_BC: broken chloroplasts of Arabidopsis; At_total: total RNA samples extracted from Arabidopsis. extension to 3’ ends (Figure 9C). Pre-2 was 77 nt in length, with a 13 nt extension outer of 3’ ends, but with a 58 nt internal deletion. According to the secondary structure deduced from E. coli (Figure 9A), Pre-1 and Pre-2 are the putative precursors of csR-5sr-1. Relationship between csRNAs and the putative targets To detect the functions of csRNAs, we searched for the transcripts of the coding genes that are complemented by the csRNAs responsive to heat stress. csR-mYCF-8, a csRNA family of Chinese cabbage (Additional File 9) that declined under heat treatment (Figure 10A), per- fectly matched to the chloroplast ycf1 gene. Using adja- cent sequences as primers, the real-time PCR showed that expression of gene dramatically increased in the HT seedlings compared with those in the MT seedlings (Figure 10B), implying a silencing role of csR-mYCF-8 csRNAs in expression of ycf1 gene. Similarly, atpE was a putative target of csR-mATP-2, which was up-regulated in the HT seedlings, just opposite to the reduced abun- dance of the csR-mATP-2 csRNAs (Figure 10). Other than ycf1 and atpE, rpoA and psbM were other two