Wang et al. BMC Genomics 2011, 12:289 http://www.biomedcentral.com/1471-2164/12/289 responsive csRNA is affected. The change in abundance of some csRNAs affects the putative target genes, which may play an important role in plant resistance to heat stress. Plant chloroplasts are closely related to leaf develop- ment, while the latter is regulated by many miRNAs and small RNAs [43-46]. However, it remains to be eluci- dated how the novel csRNAs are cooperating with the canonical miRNAs in leaf development after exposure to high temperatures and if they are affecting each other. To address the roles of these heat-responsive csRNAs in plant heat resistance, a study has been planned to com- pare the gene expression patterns of heat-resistant culti- vars of Chinese cabbage with the heat-sensitive genotypes. This study could provide insight into under- standing the molecular mechanism behind csRNA- mediated heat resistance and a fast and efficient way to improve the heat resistance of Chinese cabbage and other important crops. Conclusion The chloroplast is an important organelle that contains a plenty of small RNAs. Our results show that many members of csRNA families were highly sensitive to heat stress. Some csRNAs respond to heat stress by silencing target genes. We suggest that proper tempera- ture is important for production of chloroplast small RNA, which are associated with plant resistance to heat stress. Methods Plant materials and growth conditions Wu-11, a heat-sensitive inbred line of non-heading Chi- nese cabbage (B. rapa ssp. chinensis), was used in this study, unless indicated otherwise. The seeds were sown in pots and germinated in an incubator. After one week, the seedlings were transplanted to the growth chambers in Phytotron of SIPPE (Shanghai Institute of Plant Phy- siology and Ecology) and grown at 22°C with 16 h of light per day. Three-week-old seedlings were autoclaved at 46°C (HT, high temperature) and 22°C (MT, moder- ate temperature) in two water bath kettles, respectively, for one hour. After heat treatment, some of the seed- lings were used for RNA isolation while the rest were transferred to growth chambers for further growth. The seeds of Arabidopsis thaliana were surface-steri- lized in 70% ethanol for 1 min, followed by 0.1% HgCl2 for 10 min, washed four times in sterile distilled water and plated in molten 0.1% water agar on top of solid 1% sugar MS0 medium. Plates were sealed with Parafilm, incubated at 4°C in darkness for 2-3 days and then moved to a growth chamber at 22°C with 16 h of light per day. Page 12 of 14 RNA extraction and quantity detection After heat treatment, the above-ground portions of the seedlings were harvested, quick-frozen immediately with liquid nitrogen and stored at -80°C. Each treatment comprised 10 seedling samples that were divided into two replicates. Total RNAs were extracted using TRIzol Reagent (Invitrogen). RNA concentration was measured by Eppendorf Biophotometer 6121. DNA was removed by digestion using TURBO DNA-free kit (Ambion). Chloroplast isolation Chloroplasts were isolated from seedlings using a Per- coll (GE, 17089101) gradient-based method [47]. The intactness of chloroplast was estimated by micro- scopic examination (Olympus BX51 wide-field micro- scope with differential interference contrast) and Hill reaction [48]. Small RNA deep sequencing and sequence analysis Chinese cabbage and Arabidopsis total RNA samples were sent to Keygene N.V., Wageningen, the Nether- lands and Zhejiang-California Nanosystems Institute, China respectively, where the samples were prepared and sequenced on Illumina GAII sequencer, according to the manufacturer’s protocol. The adaptor sequences in Illumina sequencing reads were removed using “vec- torship” in the EMBOSS package. The small RNA with length of 9~36 nt were mapped to chloroplast genomes (GenBank accession number: DQ231548) and nuclear genomic sequences (BRAD, Brassica Database, http:// brassicadb.org/brad/) of Chinese cabbage (Brassica rapa ssp. chinensis) and nuclear, chloroplast, mitochondrial genomes of Arabidopsis (http://www.arabidopsis.org). All data from different samples were normalized before comparison analysis. Northern blotting analysis RNA (20 μg) was fractionated on a 19% denaturing poly-acrylamide gel (PAGE), transferred to a Hybond- XL membrane (Amersham biosciences-GE healthcare) by capillary transfer using 20 × SSC buffer, and fixed by UV-crosslinking. Pre-hybridization was carried out at 42°C for 2 × 30 min using PrefectHyb Hybridization Buffer (Sigma). Oligo-nucleotide DNA probes, were labeled with g32P ATP (5000Ci/mmol) using a T4 poly- nucleotide kinase (Roche). Hybridization was per- formed overnight at 42°C. Sizes of csRNA molecules were estimated using oligo-nucleotide DNA/RNA sense-probes with sequences same with csRNAs. In addition to equivalent RNA loading (20 μg detected by Eppendorf Biophotometer 6121), U6 spliceosomal RNA was used as an RNA loading control for total RNA sample.