title: IOLR_Cherax quadricarinatus_4.8K_v1.0
status: Public on Jan 30 2007
contact.city: Haifa
contact.first: Moshe
contact.last: Tom
contact.email: tom@ocean.org.il
contact.address: P.O.B. 8030
contact.department: Marine Biology and Biotechnology
contact.country: Israel
contact.web_link: http://www.ocean.org.il/
contact.institute: Israel Oceanographic and Limnological Research
contact.phone: 972-4-8565-257
organism: Cherax quadricarinatus
description: The array is printed on a 2.5 x 7.5 cm glass (GAPS II slides, Corning) on an area less than 6 x 2 cm. the grid system includes 4 x 12 sub-arrays, each containing 15 raws and 14 columns, a total of 10080 spots.
manufacture_protocol: X Organ – Sinus Gland complex was bi-laterally extirpated from several C. quadricarinatus males, followed after 13 or 14 days post-extirpation by sacrifice of premolted males, monitored by their gastrolith size, maxillar setae appearance and in some individuals the presence of new cuticle under the old one. Several hepatopancreatic lobes and hypodermis layered beneath the carapace and muscle tissue were dissected as well as the entire Y organ pair and RNA was extracted from the dissected tissues. The muscle served as a reference organ, whereas the molt-related hepatopancreas, epidermis and Y-organs were the target organs. The cDNA assemblage was constructed in two stages: (1) SMART (Switching Mechanism At 5' end of the RNA Transcript), aimed at amplification of reverse transcribed cDNAs using a universal primer, linked to both ends of the template cDNA during the reverse transcription stage (Clontech, manufacturer instructions). (2) SSH (Subtractive-Suppression Hybridization method; Diatchenko et al. 1999; PCR-select, Clontech), aimed at T-A cloning of a roughly equally represented cDNAs from the target organs into an expression plasmid (pGEM-Teasy, Promega). biased towards genes differentially expressed in the target tissues in comparison to the reference one. The cloned cDNAs were transfected into E. coli, and 4800 bacterial colonies were isolated, amplified and frozen at -70C. A subset of the clones were successfully sequenced and assembled by the CAP3 software (Huang and Madan, 1999), to identify unique sequences. All the 4800 clones were amplified by standard PCR from 1 µl bacterial culture templates (5’ at 95C; 32 cycles of 0.5’ at 95C, 0.5’ at 68C and 1.5’ at 72C; a final incubation of 4’ at 72C; GeneAmp 5700, ABI). PCR products were precipitated from the PCR solution by one volume of isopropanol at room temperature. The dry DNA was dissolved in printing buffer at final concentration of 50% DMSO and 0.3 x SSC and was printed on a glass slide (GAPS II slides, Corning; MGII robot-printer, BioRobotics) in duplicates. The amplification efficiency was tested by running the PCR products on a 1% agarose gel concurrent with a DNA mass ladder. The printing pattern included two complete and separate sets of the transcriptome, in the upper and lower part of the slide, respectively.
  Refernces:
  Diatchenko, L., Lukyanov, S., Lau, Y.F.C. Siebert, P.D. (1999) uppression subtractive hybridization: A versatile method for identifying differentially expressed genes. In: cDNA Preparation and characterization, Meth. Enzymol. 303: 349-380.
  Huang, X. and Madan, A. (1999) CAP3: A DNA sequence assembly program. Gen Res 9: 868-877.