The protocol for designing the array is described in detail in Crump, D, Werry, K, Veldhoen, N, van Aggelen, G, and Helbing, CC (2002) Environmental Health Perspectives 110: 1199-1205. Preparation of RNA. Total RNA was obtained from preserved tadpole tissue using TRIzol reagent as described by the manufacturer (Invitrogen Canada Inc., Burlington, Ontario, Canada). We subsequently resuspended isolated RNA in RNase-free water and stored it at –70°C. Poly(A)+ RNA was isolated from total RNA using the Oligotex mRNA isolation mini kit (Qiagen Inc., Mississauga, Ontario, Canada). Array target sequence selection and primer design. The frog MAGEX (multispecies analysis of gene expression) cDNA array was designed using Xenopus and Rana complete cDNA information obtained from GenBank (GenBank 2001). The abundance of Xenopus cDNA sequences compared to Rana sequences on the array reflects their relative abundance in GenBank. The cDNA sequences chosen encode structurally and functionally important products involved in regulation of amphibian development (embryogenesis and metamorphosis) as well as basal cell metabolism (ViagenX Biotech, Inc. 2002). We selected X. laevis ribosomal L8 (GenBank accession no. U00920) cDNA for normalization, as its mRNA transcript levels remain relatively constant between different developmental stages (Shi and Liang 1994). We included 420 cDNA sequences on the array, of which 390 are X. laevis and 30 are R. catesbeiana in origin. All primers were designed with Primer Premier V4.1 software (Premier Biosoft International, Palo Alto, CA, USA) and synthesized by AlphaDNA (Montreal, Québec, Canada). Primers of 18 to 20 base pairs with an optimal annealing temperature between 50° and 55°C were designed to amplify sequences of between 450 and 550 base pairs within each target cDNA. Primer pairs were combined and diluted to a final concentration of 10 μM. Total cDNA preparation. We prepared individual cDNA target sequences which form the basis of the array clone bank from total RNA isolated from untreated Xenopus tadpoles (NF stages 47 and 58; Nieuwkoop and Faber 1956) and oocyte tissues and Rana tadpoles (stage XV; Taylor and Kollros 1946) using TRIzol reagent as described by the manufacturer (Invitrogen). Total RNA was annealed with 500 ng random hexamer oligonucleotide (Amersham Biosciences, Baie d’Urfe, Québec, Canada) and cDNA was generated using Superscript II RNase H-reverse transcriptase as described by the manufacturer (Invitrogen). The 20-μL reaction was incubated at 42°C for 2 hr and diluted 20-fold before DNA amplification. Amplification, cloning, and verification of target cDNAs. Each 50-μl DNA amplification reaction contained polymerase chain reaction buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2), 200 μmol dNTP, 20 pmol of each gene specific primer, 1 μL of a 20-fold dilution of X. laevis or R. catesbeiana total cDNA, and 2.5 U Taq DNA polymerase (Invitrogen). Amplification was carried out in a DNA Thermal Cycler 480 (Perkin-Elmer, Wellesley, MA, USA). The thermocycle program included a denaturation step at 94°C (7 min); 45 cycles of 94°C (1 min), 52°C (1 min), and 72°C (1 min); and a final elongation step at 72°C (7 min). Amplified DNA products were separated on a 1.5% agarose gel and visualized by ethidium bromide staining on a ChemiImager 4000 (Alpha Innotech Corp., San Leandro, CA, USA). We excised DNA bands within the correct size range (450–550 base pairs) from the gel and extracted them by freeze-thaw centrifugation (Smith 1980). We confirmed purified samples by electrophoresis on a 1.5% agarose gel. Polymerase chain reaction products were cloned into pCR II-TOPO vector using the TOPO TA Cloning Kit (Invitrogen). We purified plasmid DNA using the QIAprep Spin Miniprep Kit (Qiagen) and confirmed the presence of insert sequence by restriction analysis using EcoRI (Amersham Biosciences). Cloned cDNA was sequenced and compared by BLAST to sequences available in GenBank. Clones that matched the correct target sequence were included in the plasmid clone bank. Preparation of cDNA fragments for spotting on the array. We prepared specific cDNA fragments for the frog MAGEX cDNA array using the plasmid clone bank. Individual amplification reactions were assembled in 96- well amplification plates (VWR International, Mississauga, Ontario, Canada). Each 100-μl reaction was similar to that described above, except that the target DNA comprised 1 ng of each plasmid clone, and 1 U AmpliTaq polymerase (Applied Biosystems, Foster City, CA, USA) was used. Amplification was carried out in an MJ Research Gradient Cycler PTC-255 (MJ Research, San Francisco, CA, USA). The thermocycle program included a denaturation step at 95°C (2 min); 35 cycles of 95°C (30 sec), 52°C (30 sec), and 72°C (1 min); and a final elongation step at 72°C (10 min). We separated amplified DNA products by electrophoresis on a 1.5% agarose gel and purified them using the QIAquick PCR purification kit and the Qiagen Biorobot 3000. Purified cDNA fragments were resuspended in 3SSC (20SSC contains 3 M NaCl, 0.3 M Na citrate, pH 7.0) to a final concentration of 0.5 μg/μL. The prepared cDNA was then loaded into 384-well plates (VWR International), and 90 nL/spot was spotted in duplicate on Biodyne B positively charged nylon membranes (PALL Gelman Laboratory, Ann Arbor, MI, USA) using the Biorobotics Total Array System (Biorobotics Ltd., Woburn, MA, USA). The spotted membranes were UV-crosslinked and stored at –20°C.
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