Wang et al. BMC Genomics 2011, 12:289 http://www.biomedcentral.com/1471-2164/12/289 Quantitative real-time reverse transcription PCR 10 mg of each Chinese cabbage total RNA sample were treated twice with 4 U of TURBO DNase (Ambion) for 30 min at 37°C. For cDNA synthesis, 200 ng RNA were reverse transcribed using SuperScript™ First-Strand Synthesis System with random hexamers (Invitrogen) according to manufacturer’s recommendations. A MyiQ2 real-time PCR detection system (Bio-rad) was used for qPCR (2 min at 50°C, 10 min at 95°C, 40 × (15 s at 95 °C, 1 min at 58°C)) with the Power SYBR Green kit (Applied Biosystems; 10 μl master mix, 5 μl cDNA (diluted 1: 10 for chloroplast atpE, psbM, rpoA and ycf1; and 1: 5 for Chinese cabbage UBQ5), 1 μl of forward and reverse primer (1 μM each), 3 μl H2O; atpE pri- mers: S-tttgctgagcttcttgtgga, A- cgaatcgaattgtttgggat; psbM primer: S-ggaacgagaatgaagagtgc, A-tcccgagatattc- caaagaa; rpoA primer: S-agattctgggaggcaattct, A-aag- cacttcatcaagcctcc; ycf1 primers: S-accaatccaacccatttcat, A-ccaagttcaatgttagccaga; UBQ5 primers: S-tccgtccaccttg- tagaactg, A-tgaaaaccctaacggggaaa). At least four biologi- cal replications were detected for each gene. All reactions were done in triplicate, and for each reaction a ‘no RT’ control was included. Data analysis was per- formed with the iCycler IQ real-time detection system (Bio-Rad), with automatic Ct and baseline setting and UBQ5 mRNA concentrations as internal control. Rela- tive transcript quantities were calculated with the ΔΔCt method. Circular reverse transcription PCR (cRT-PCR) The precise 5’ and 3’ ends of the csRNA precursors were determined by circular RT-PCR. The DNase treated chloroplast RNAs were incubated with 40U of T4 RNA ligase (New England Biolabs) according to manufacturer’s protocol, which could generate circular RNAs with the 5’ and 3’ ends joined together. After phenol-chloroform extraction, single-strand cDNAs were synthesized using Revertra Ace (TRT-101, Toyobo) and specific reverse primer. The sequences of the reverse transcript products were complemen- tary to the small RNA precursors, with breakpoints at the ends. PCR amplification were then carried out using a forward primer (cRT Primer 1: gctcgacgccag- gatg) as same sequence as csR-5sr-1, and a reverse primer (cRT Primer 2: gtgttaagcttttcat) which comple- ment to the DNA sequence following the binding site of csRNA (indicated in Figure 9). PCR amplification consisted of 30 cycles of 30s duration at 94°C, 30s at 48°C and 30s at 72°C. All cRT-PCR products were purified by MagExtractor (NPK-600, Toyobo), and then ligased into pMD18-T vector (D101A, Takara) for sequencing. Page 13 of 14 Additional material Additional file 1: Abundance of sequenced small RNAs matched to genomes of Arabidopsis thaliana (Landsberg erecta ecotype). Additional file 2: Comparison analyses of Arabidopsis chloroplast- related small RNAs (Related) and chloroplast-specific small RNAs (Specific). Additional file 3: RNA origin analysis of Chinese cabbage csRNAs. Additional file 4: Size distribution of csRNAs in the HT seedlings. Additional file 5: csRNAs of csR-5sr-1 family. Additional file 6: The position analysis of the 3’ ends of rRNA- derived csRNAs of Chinese cabbage. Additional file 7: The csRNAs that were reduced in the HT lines (less than 20%). Additional file 8: The csRNAs that were increased in the HT lines with 5 folds. Additional file 9: csRNAs detected by microRNA microarray analysis. Additional file 10: rRNA-derived csRNAs were drastically reduced in the HT seedlings. Additional file 11: The predicted structures of some abundant csRNAs from 4.5S and 16S rRNAs, and from tRNA-Asp. Abbreviations cRT-PCR: circular semi-quantitative reverse transcription polymerase chain reaction; csRNA: chloroplast small RNA; DDM1: decrease in DNA methylation 1; dsRNA: double-stranded RNAs; HT: high temperature; igRNA: intergenic RNA; MET1: cytosine methyltransferase mutant 1; mRNA: messenger RNA; MT: moderate temperature; ncRNA: non-coding RNA; nt: nucleotide; rRNA: ribosomal RNA; siRNA: small interfering RNA; snoRNA: small nucleolar RNAs; tRNA: transfer RNA. Acknowledgements and Funding We thank Dr JR Huang and Y Sun (SIPPE, China) for their help on intact chloroplast isolation. This work was supported by grants from the Natural Science Foundation of China (30730053). Author details 1National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China. 2Keygene, N.V., Agro Business Park 90, 6708 PW Wageningen, The Netherlands. 3James D. Watson Institute of Genome Sciences, Zhejiang University, 268 Kaixuan Road, Hangzhou 310012, China. 4T-Life Research Center Fudan University, Shanghai 200433, China. Authors’ contributions The work presented here was carried out in collaboration between all authors. HYK defined the research theme and directed the experiments. WL carried out the chloroplast isolating, small RNAs, precursors and target genes detecting, some of the bioinformatics analyses, and participated in manuscript writing. YX and LYZ constructed the small RNA libraries and performed most of the bioinformatics analyses. WH, PM and DRM co-worked on the small RNA sequencing. HYK, PM, YX and WH also revised this paper. All authors have contributed to, seen and approved the manuscript. Received: 20 January 2011 Accepted: 3 June 2011 Published: 3 June 2011 References 1. Hüttenhofer A, Schattner P, Polacek N: Non-coding RNAs: hope or hype? Trends Genet 2005, 21:289-297. 2. Hannon GJ: RNA interference. Nature 2002, 418:244-251.