title: Black tiger shrimp subtractive hybridization cDNA array
status: Public on Jan 03 2008
contact.city: Charleston
contact.first: Enrique
contact.last: de la Vega
contact.email: delavega@musc.edu
contact.state: SC
contact.address: 331 Fort Johnson Road
contact.department: Biochemistry
contact.country: USA
contact.institute: Medical University of South Carolina
contact.phone: 1-843-7628820
organism: Penaeus monodon
manufacture_protocol: Construction of cDNA libraries
  Suppression subtractive hybridization (SSH) (Diatchenko et al. 1996), was used to generate cDNA libraries enriched for genes that are regulated under different types of environmental stress exposure. Double stranded cDNA was prepared from 2 µg poly A RNA extracted from shrimp haemocytes (pool of three shrimp), digested with Rsa I, and tester and driver cDNAS generated for treatments and controls using the PCR-Select cDNA Subtraction Kit (Clontech) according to the manufacturer’s instructions. The PCR-amplified cDNA fragments generated by SSH were then ligated into the plasmid pGEM-T Easy (Promega) and used to transform High Efficiency JM109 competent cells (Promega) according to the manufacturer’s instructions. Six different SSH cDNA libraries were constructed enriching for both up and down regulated genes during osmotic, hypoxic and hyperthermic environmental stress exposure. A total of 1248 random recombinant colonies were hand-picked, cored and further amplified in 96 well plates with LB ampicillin.
  A full-length enriched cDNA haemocyte library in λ TriplEx2 was also constructed from haemocytes of a single hyperthermic stressed shrimp using the SMART cDNA library construction kit (Clontech) following manufacturer’s instructions for long distance PCR. The cDNA ligated into λ TriplEx2 arms was packaged using the Gigapack III Gold ® packaging system (Stratagene) and the phage used to infect E. coli, strain XL1-blue cells. A total of 2688 random recombinant plaques were cored and the phage eluted in SM buffer.
  PCR amplification and construction of cDNA microarray
  The insert from each of the 3936 cDNA fragments from the SSH libraries and the non-subtracted haemocyte library was PCR-amplified using either pTriplEX2 sequencing forward and reverse primers (Clontech) for clones from the pTriplEx2 cDNA library or SSH nested primers 1 and 2R (Clontech) for clones from the SSH libraries. PCR products were assessed by agarose gel electrophoresis (Holloway et al. 2003), isopropanol precipitated, washed with 70% ethanol and resuspended in 60 μl of 4X SSC, 0.1% sarkosyl.  The 4032 DNA elements composed of PCR products from the different cDNA libraries and a series of controls were spotted in duplicate onto polylysine-coated glass slides using a GMS 417 Arrayer robot (Genetic Microsystems). Post-processing of the slides was accomplished as previously described (Woods et al. 2004 Development 131(12): 2921-2933.).