Alpha imager imaging system

The semi-quantitative RT-PCR, for the amplification of GFP and suppressor genes (RTBV ORF-IV, RTBV ORF-I, and RTSV ORF-protease), was carried out using gene specific primers at initial sample denaturation at 95 °C for 5 min, followed by 25 cycles of strand separation at 94 °C for 1 min, annealing at 56 °C for 30 s, and extension at 72 °C for 30 s. The program was extended for 7 min at 72 °C. The tobacco 18S gene was used as a constitutive internal standard to evaluate cDNA content. The amplification products were analyzed on 0.8% agarose gel. The band intensities were quantified using the Alpha Imager Imaging System (Alpha Innotech, San Leandro, CA, USA).

A native gel-based assay for examining SOD activity was carried out according to a previously described method [48 (link)] with slight modifications. Cells were sonicated in a 50 mM potassium phosphate buffer (pH 7.8). Cell protein (20 µg/lane) was run on a native riboflavin gel comprising a 5% stacking gel (pH 6.8) and a 12% running gel (pH 8.8) at 4 °C. To visualize SOD activity, gels were first soaked in 2.43 mM nitro blue tetrazolium (Wako Pure Chemical Industries, Osaka, Japan) in deionized water for 20 min and then in 56 nM riboflavin (Wako Pure Chemical Industries, Ltd., Osaka, Japan) with 28 mM N,N,N′,N′-tetramethylethylenediamine (Sigma Aldrich, St. Louis, MO, USA) in a 50 mM potassium phosphate buffer (pH 7.8) for 15 min in the dark. Gels were washed with deionized water and illuminated under fluorescent light until clear zones of SOD activity were evident. The images were recorded and MnSOD bands were quantified using the AlphaImager imaging system (Alpha Innotech, San Leandro, CA, USA). MnSOD activity was assessed by examining the band density. The MnSOD activity of vector-only-transfected control cells was normalized to a value of 1.0 and relative MnSOD activities in other cells were calculated. Results were calculated as the mean of the integrated density from five independent runs.

The purified PCR product was loaded onto 8% (w/v) polyacrylamide gels (ratio of acrylamide to bisacrylamide, 37.5:1). The denaturing gradients used ranged from 30 to 70% denaturant [100% denaturant contained 7 M urea and 40% (v/v) formamide]. The gels were electrophoresed using a D-Code instrument (Bio-Rad) in 1 × TAE buffer (40 mM Tris-acetate, 1 mM Na-EDTA, pH 8.0) running at 30 V for 15 min first and then 130 V for 4.5 h at 60°C.
After electrophoresis, the gel was stained with ethidium bromide (10 μg/ml) for 15 min, and images were captured using an Alpha-Imager Imaging System with AlphaEase FC image software 4.1.0 (Alpha Innotech).
Each visible band on the DGGE gels containing the PAH-degrading consortia was excised manually, and the DNA was extracted using the method described by Muyzer et al. (1993) (link). Using 5 μl of the extracted DNA as template, PCR was carried out to amplify target DNA for cloning. Positive PCR products were purified using the E.Z.N.A Cycle-Pure Kit (OMEGA Bio-tek, USA) and cloned into the pMD19-T Vector (Takara). After being confirmed using another DGGE gel, they were sequenced with an ABI model 3730 DNA sequencer (Invitrogen Shanghai).