Bao and Xu BMC Genomics 2011, 12:224 http://www.biomedcentral.com/1471-2164/12/224 synthesized using 1 μg of total RNA at 37°C for 1 h, with an M-MLV reverse transcription system (Promega, Madi- son, USA). The primers used to identify of differentially expressed transcripts by RT-PCR are presented in Addi- tional File 4. The PCR reactions were subjected to 22–26 cycles consisting of 94°C for 30 s; 55°C for 30 s; 72°C for 1 min. Actin was used as an internal standard. Northern blot hybridization Total RNA (25 μg) from the brain of day 1–2 diapause- and nondiapause-destined pupae was separated on a 1.2% agarose gel containing 0.22 mol/L formaldehyde, and transferred to a nylon membrane (Hybond N+, Amersham). Probes for hybridization were labeled with [a-32P]-dCTP using the Random Primer Labeling kit (Takara). After prehybridization for 4 h in 5× SSPE (1 × SSPE = 180 mM NaCl, 10 mM sodium phosphate, pH 7.7, 1 mM EDTA) containing 50% formamide, 5× Den- hardt’s solution, 0.1% SDS, and 100 μg/mL salmon sperm DNA, the radiolabeled probe was added and hybridization was conducted overnight at 42°C [48]. After hybridization, the membrane was washed in 0.2× SSPE at 42°C three times and exposed to X-ray film (Kodak) overnight at -70°C. Polyclonal antibody generation and western blot analysis The ORFs of four genes (SUMO, MnSOD, sericotropin and TCTP) were amplified by PCR, using primers that contained restriction sites. The PCR product was digested by the appropriate restricted enzymes, then purified and subcloned into the pET28a vector. The recombinant pET plasmid was transfected into BL21 cells and induced by IPTG. The E. coli pellet was solubi- lized in 6 M urea in 50 mM Tris-HCl buffer, pH 8.0, followed by Ni-NTA column purification. Purified recombinant proteins were used to generate polyclonal antibodies in rabbit. Proteins for western blotting were extracted from the brain and SG or the brain–SG complexes of pupae, quantified by the Bradford method [49], and stored at -80°C. Protein (20 μg) was separated by 15% SDS-PAGE and transferred onto an Immobilon-P Transfer mem- brane (Millipore). The immuno-reactivity was tested with antiserum, and then incubated with goat anti-rabbit IgG (Thermo), and protein was detected using the Novex Chemiluminescent Substrates (Invitrogen). Additional material Additional file 1: Table S1 Checklist of known genes in the two libraries. (A) Checklist of the sequences of known genes in the F library. (B) Checklist of the sequences those were homologous to known genes in the R library. All transcripts were compared to the sequences in GenBank using Blastx. Sequence description, minimum E-value and similarity are shown in the table. Page 13 of 14 Additional file 2: Table S2 Comparison of the two libraries according to Gene Ontology criteria (Biological Process). Additional file 3: Figure S1 Classification of SSH ESTs according to Gene Ontology Criteria (Molecular Function and Cellular Component).(A) Classification of SSH ESTs according to Gene Ontology criteria (Molecular Function). Gene Ontology analysis was carried out on the transcripts isolated from the two SSH libraries by Blast2GO program. The molecular Function combined graph was made based on ontology level 2. (B) Classification of SSH ESTs according to Gene Ontology criteria (Cellular Component). The cellular Component combined graph was made based on ontology level 2 by Blast2GO. Additional file 4: Table S3 Primer sequences used in PCR. Acknowledgements This study was supported by a Grant-in-Aid for the Natural Scientific Foundation (30730014) from the National Natural Science Foundation of China, and the Major State Basic Research Developmental Program (2006CB102001) from the Ministry of Science and Technology of China. Authors’ contributions WHX designed the research project, BB performed experiments. BB and WHX wrote the paper, and approved the final manuscript. Received: 9 November 2010 Accepted: 11 May 2011 Published: 11 May 2011 References 1. Tauber MJ, Tauber CA, Masaki S: Seasonal adaptations of insect. Oxford University Press, New York; 1986. 2. Denlinger DL: Hormone control of diapause. In Comprehensive Insect Physiology. Edited by: Kerkut GA, Gilbert LI. Biochemistry and Pharmacology. Pergamon Press, Oxford; 1985:353-412. 3. Kostal V: Eco-physiological phase of insect diapause. J Insect Physiol 2006, 52(2):113-127. 4. 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