2.4. DISCUSSION 4.2.4. SSH Protocol The PCR-Select cDNA subtraction kit (Clontech, USA) was used for SSH, according to the manufacturer’s instructions. The GeneAmp PCR System 9700 thermal cycler (Perkin Elmer Applied Biosystems, USA) was used for all incubation steps (unless indicated otherwise) and PCR reactions. 4.2.4.1. First-strand cDNA synthesis To generate first-strand cDNA from each tester and driver mRNA samples, as well as the control poly A+ RNA, the following reagents were combined in a 0.5 ml microcentrifuge tube: 4 µl mRNA (2 µg) (2 µl control poly A+ RNA) and 1 µl cDNA synthesis primer (10 µM). Contents were mixed thoroughly and incubated at 70°C for 2 min in the GeneAmp PCR System 9700 thermal cycler. Tubes were cooled on ice for 2 min, briefly centrifuged and the following reagents added: 2 µl 5× first-strand buffer [250 mM Tris.HCl (pH 8.5), 40 mM MgCl2, 150 mM KCl, and 5 mM DTT], 1 µl dNTP mix (10 mM each), 1 µl sterile H2O and 1 µl AMV reverse transcriptase (20 units/µl). Contents were gently vortexed and briefly centrifuged before incubation at 42°C for 1.5 h in an air incubator. First- strand cDNA synthesis was terminated by placing the tubes on ice and second- strand cDNA synthesis was commenced immediately. 4.2.4.2. Second-strand cDNA synthesis The following components were added to the first-strand tester, driver and control cDNAs: 48.4 µl sterile H2O, 16 µl 5× second-strand buffer [500 mM KCL, 50 mM NH3SO4, 25 mM MgCl2, 0.75 mM β-NAD, 100 mM Tris.HCl (pH 7.5), and 0.25 mg/ml BSA], dNTP mix (10 mM), and 20× second-strand enzyme cocktail (6 U/µl DNA polymerase I, 0.25 U/µl RNase H, and 1.2 U/µl E. coli DNA ligase). Contents were briefly centrifuged before incubation at 16°C for 2 h in a thermal cycler. Two µl T4 DNA polymerase was added and mixed well before incubation at 16°C for 30 min in a thermal cycler. Four µl 20× EDTA/glycogen mix (0.2 M EDTA, and 1 mg/ml glycogen) was added to terminate second-strand synthesis. One hundred µl phenol:chloroform:isoamyl alcohol (25:24:1) was added, vortexed thoroughly and centrifuged at 14 000 rpm for 10 min at RT. The upper layer was placed in a clean tube and 100 µl chloroform:isoamyl alcohol (24:1) was added. Tubes were vortexed thoroughly and centrifuged at 14 000 rpm for 10 min at RT. The upper layer was placed in a clean tube and 40 µl 4 M CH3COONH4 and 300 µl 95% ethanol was added. Tubes were vortexed thoroughly and centrifuged at 14 000 rpm for 20 min at RT. Supernatants were carefully removed and pellets were overlaid with 500 µl 80% ethanol and centrifuged at 14 000 rpm for 10 min at RT. Supernatants were carefully removed and pellets were air-dried and dissolved in 50 µl sterile H2O and stored at -20°C until RsaI digestion. 4.2.4.3. RsaI digestion In order to create smaller blunt-end tester and driver cDNA fragments, the generated cDNAs were digested with RsaI. The following reagents were added to 43.5 µl of each tester, driver and control second-strand cDNAs: 5 µl 10× RsaI restriction buffer [100 mM Bis Tris propane-HCl (pH 7.0), 100 mM MgCl2, and 1 mM DTT] and 1.5 µl RsaI (10 U/µl). Tubes were mixed by vortexing and briefly centrifuged before incubation at 37°C for 1.5 h. Two and a half µl of 20× EDTA/glycogen mix (0.2 M EDTA, and 1 mg/ml glycogen) was added to terminate second-strand synthesis. Fifty µl phenol:chloroform:isoamyl alcohol (25:24:1) was added, vortexed thoroughly and centrifuged at 14 000 rpm for 10 min at RT. The upper layer was placed in a clean tube and 50 µl chloroform:isoamyl alcohol (24:1) was added. Tubes were vortexed thoroughly and centrifuged at 14 000 rpm for 10 min at RT. The upper layer was placed in a clean tube and 25 µl 4 M NH4OAc and 187.5 µl 95% ethanol were added. Tubes were vortexed thoroughly and centrifuged at 14 000 rpm for 20 min at RT. Supernatants were removed and pellets were overlaid with 200 µl 80% ethanol and centrifuged at 14 000 rpm for 5 min at RT. Supernatants were carefully removed and pellets were air dried and dissolved in 5.5 µl sterile H2O and stored at -20°C. These samples served as experimental driver and control driver cDNAs. 4.2.4.4. Adaptor ligation Tester and control tester cDNAs were ligated with adaptors for forward and control reactions. One µl RsaI-digested experimental tester cDNA was diluted with 5 µl sterile H2O and control cDNA was diluted with diluted φX174/HaeIII control DNA according to the manual instructions, prior to adaptor ligation. A ligation master mix was prepared by combining the following reagents: 3 µl sterile H2O, 2 µl 5× ligation buffer [250 mM Tris.HCl (pH 7.8), 50 mM MgCl2, 10 mM DTT, and 0.25 mg/ml BSA] and 1 µl T4 DNA ligase (400 U/µl). Ligation reagents for each adaptor were added to a 0.5 ml microcentrifuge tube in the following order: 2 µl diluted tester cDNA, 2 µl of adaptors 1 or 2R (10 µM), respectively, and 6 µl master mix. Contents were mixed thoroughly by pipetting. Two µl of each tester-adaptor mixture was mixed in a clean microcentrifuge tube to serve as the unsubtracted tester control. Tubes were briefly centrifuged and incubated O/N at 16°C. One µl EDTA/glycogen mix was added to terminate the ligation reaction and samples were heated at 72°C for 5 min to inactivate the ligase. One µl of the completed unsubtracted tester control was removed and diluted in 1 ml sterile H2O for PCR analysis. All samples were stored at -20°C. Ligation efficiency analysis of the control cDNA was performed prior to the hybridization steps as described in the PCR-Select cDNA subtraction manual. 4.2.4.5. First hybridization In the first hybridization, excess driver cDNA was added to each adapter-ligated tester cDNA to enrich for differentially expressed sequences. Hybridization reagents were combined in 0.5 ml microcentrifuge tubes for each experimental and control subtractions in the following order: 1.5 µl RsaI-digested driver cDNA, 1.5 µl adaptor 1-ligated or adaptor 2R-ligated tester, respectively, and 1 µl 4× hybridization buffer. Samples were overlaid with one drop mineral oil and centrifuged briefly before incubation at 98°C for 1.5 min in the thermal cycler. Samples were then incubated at 68°C for 8 h. 4.2.4.6. Second hybridization The two samples from the first hybridization were mixed and freshly denatured driver cDNA was added to further enrich for differentially expressed sequences. Hybridization reagents were combined in 0.5 ml microcentrifuge tubes: 1 µl driver cDNA, 1 µl 4× hybridization buffer, and 2 µl sterile H2O. For each experimental and control tester cDNA, 1 µl of this mixture was placed in a clean 0.5 ml microcentrifuge tube, overlaid with mineral oil and incubated at 98°C for 1.5 min in a thermal cycler. Freshly denatured driver cDNA was then mixed with the two hybridization samples from the first hybridization according to the manufacturer’s instructions. Contents were briefly centrifuged and incubated O/N at 68°C. Two hundred µl dilution buffer [20 mM HEPES (pH 6.6), 20 mM NaCl, and 0.2 mM EDTA (pH 8.0)] was added and mixed by pipetting before incubation at 68°C for 7 min in a thermal cycler. All samples were stored at -20°C until further use. Subtraction efficiency was determined by using PCR analysis with G3PDH primers according to the manufacturer’s instructions. 4.2.4.7. PCR amplification Differentially expressed cDNAs were selectively amplified during the two PCR reactions as described below. One µl of each diluted cDNA (each subtracted sample and the corresponding unsubtracted tester control) and control subtracted cDNA were aliquoted into PCR tubes and the following PCR reagents were added to each tube: 19.5 µl sterile H2O, 2.5 µl 10× PCR reaction buffer, 0.5 µl dNTP mix (10 mM), 1 µl PCR primer 1 (10 µM), and 0.5 µl 50× Advantage cDNA Polymerase Mix (Clontech). Contents were mixed well by vortexing, briefly centrifuged, overlaid with mineral oil and incubated at 75°C for 5 min in the thermal cycler. Primary PCR was commenced using the following cycling parameters: 94°C for 25 s, followed by 27 amplification cycles of 94°C for 10 s, 66°C for 30 s, and 72°C for 1.5 min. Eight µl from each tube was analyzed on 2% agarose/ethidium bromide gel run in 1× TAE buffer. Three µl of each primary PCR mixture was then diluted in 27 µl sterile H2O of which 1 µl was aliquoted into clean PCR tubes for secondary PCR. The following PCR reagents were added to each tube: 18.5 µl sterile H2O, 2.5 µl 10× PCR reaction buffer, 1 µl nested PCR primer 1 (10 µM), 1 µl nested PCR primer 2R (10 µM), 0.5 µl dNTP mix (10 mM), and 0.5 µl 50× Advantage cDNA Polymerase Mix (Clontech). The contents were mixed well by vortexing and briefly centrifuged. Samples were overlaid with one drop mineral oil and secondary PCR was commenced using the following cycling parameters: 12 cycles of 94°C for 10 s, 68°C for 30 s, and 72°C for 1.5 min. Eight µl from each tube was analyzed on 2% agarose/ethidium bromide gel run in 1X TAE buffer. Visual analysis of secondary PCR products revealed poor amplification. Thus, the protocol was modified in order to obtain optimal amounts of primary PCR products. This modification involved increasing the number of amplification cycles for the primary PCR from 27 to 30. The resulting amplicons were used for further analysis. All reaction products were stored at -20°C until further use. In document Comparative proteomic and genomic analysis of Flavobacterium johnsoniae-like biofilm, planktonic and agar surface-associated cells (Page 141-146)
