Moshe Tom's laboratory - Israel Oceanographic and Limnological Research, Haifa, Israel.
Manufacture protocol
Exposure to pollutants was performed after a one month acclimation period, by injecting 5 xenobiotic mixtures dissolved in a suitable vehicle, totaling 10 compounds, into 5 groups of fish at the first and third day of the experiment. The utilized compounds were: Benzo(a)pyrene, 1,1 dichloro-2.2-bis [p-chlorophenyl] ethylene (pp’-DDE), Aroclor 1254 (polychlorinated biphenyl mixture, perfluoro octanoic acid, tributyl tin chloride, Lindane, methyl mercury chloride, 4-nonylphenol, beta-estradiol and cadmium chloride. Non-injected fish served as reference group. Fish were sacrificed by decapitation at the third and the seventh day after the second injection. Livers were snap frozen in liquid nitrogen and kept at -80oC, aimed at latter RNA purification. 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 4608 bacterial colonies were isolated, amplified and frozen at -700C. All the 4608 clones were amplified by standard PCR (5’ at 950C; 32 cycles of 0.5’ at 950C, 0.5’ at 680C and 1.5’ at 720C; a final incubation of 4’ at 720C; GeneAmp 5700, ABI) using the PCR-select universal primers located at both ends of each cloned DNA fragment (see Diatchenko et al. 1999; PCR-select, Clontech). 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 adjacent duplicates. The amplification efficiency was tested by running the PCR products on a 1% agarose gel concurrent with a DNA mass ladder. Most of the clones were successfully sequenced and assembled by the CAP3 software (Huang and Madan, 1999), to identify unique sequences. All unique sequences were annotated by BLASTN, BLASTX and GO. References Diatchenko, L., Lukyanov, S., Lau, Y.F.C. and Siebert, P.D. (1999) Suppression subtractive hybridization: A versatile method for identifying differentially expressed genes. In: cDNA Preparation and Characterization, Meth Enzymol 303: 349-380. San Diego: Academic Press Inc. Huang, X. and Madan, A. (1999) CAP3: A DNA sequence assembly program. Gen Res 9: 868-877.
Support
Glass
Coating
Aminosilane
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 14 rows and 14 columns, a total of 9408 spots.
Less...
More...