Harmen J. G. van de Werken1 and Marcel R. A. Verhaart
Growth protocol
C. saccharolyticus was subcultured (overnight) 3 times on the substrate of interest in modified DSMZ 640 medium before inoculating a pH-controlled (pH = 7) 1-liter fermentor containing 4 gram substrate per liter. Cells were grown at 70 °C until mid-logarithmic phase (~OD660 = 0.3-0.4)
Extracted molecule
total RNA
Extraction protocol
RNA Isolation Protocol: Pump 50ml of cells into precooled Greiner tubes and cool down on ice. Spin down 20 min at 4600 rpm at 4 ºC. Store pellets at -80 ºC (at least overnight). Resuspend in 1 ml Trizol and incubate 5 min at room temperature. Tranfer to a new eppindoff tube and add 0.2 ml chloroform. Shake and incubate for 5 min at room temperature. Spin down for 15 min at 10.000 rpm at 4 ºC. Transfer aqueous phase to new tube. Add an equal amount of 70% EtOH. Transfer the sample to an RNeasy spin column. Close lid and centrifuge for 15 sec at 10.000 RPM. After centrifugation, carefully remove the spin column form the collection tube so that the column does not contact the flow-through. Pipet the DNase I incubation mix (10 ml DNase I stock and 70 ml RDD buffer) directly onto the RNeasy membrane and incubate at room temperature for 15 min. Pipet 350 ml RW1 into the RNeasy column, incubate 5 min. and centrifuge 15s at 10.000 RPM. Wash with 700 ml RW1 and throw away the collection tube. Take a collection new tube and wash column twice with 500 ml RPE. Centrifuge the column 1 min at 10.000 RPM. Place the column onto a new tube and elute twice with 30 ml RNase free water, incubate for 5 min at room temperature. Store at -80°C.
Label
Cy™3 dye (GE Healthcare)
Label protocol
Producing cDNA: In a sterile PCR tube add 20 ug RNA with 6 ug random primers. Then add nuclease free water so the volume is 10 uL. Incubate at 70 degree C for 10 min. Place tube on ice for 1 min. Add 8 uL of RT Master Mix (recipe below) to each tube and swirl to mix. Quick spin before incubation. RT Master Mix: 4uL 5X strand buffer, 1uL 0.1M DTT, 1uL dNTP mix (see below), 2uL superscript III Invitrogen. dNTP mix: 3uL dATP (100mM), 3uL dGTP (100mM), 3uL dCTP(100mM), 1.8uL dTTP (100mM), 2.4uL aa-dUTP(50mM) Ambion, 6.8uL nuclease free water. Incubate at 46 degree C for at least 3 hours. Hydrolyze RNA by adding 10uL 1N NaOH to each tube. To stop reaction add 10 uL 0.5M EDTA to each tube. Mix and incubate at 65 degree C for 15 min. Add 10uL 1N HCl to each tube neutralize reaction mix. Transfer samples into labeled tubes of appropriate sizes. Add five volumes PB Buffer (Qiagen) to each tube and mix well. Add 300 ul of RT product to a labeled Qiagen PCR cleanup column. Spin at 1,000 rpm for 15 sec., empty collection tube, and repeat until all of the solution has passed through the column. Complete wash One, add 750 ul of phosphate wash buffer (recipe below) to column and spin at 13,000 rpm for 1 min., empty tube. Complete wash Two, add 750 ul of phosphate wash buffer to column and spin at 13,000 rpm for 1 min., empty tube. Phosphate wash buffer: 0.1mL 1M KPO4( pH 8.5), 3.05mL autoclaved dH2O and 16.85mL 95% EtOH. Spin an additional 1 min. with the same collection tube and discard the flow through. Transfer to new 1.5 mL tube, add 60uL phosphate elution buffer to the membrane and incubate at room temp. for 1 min. Spin at 13,000 rpm. Phosphate elution buffer: 4mM KPO4. Repeat phosphate elution into same tube using 60uL. Fluorescent Labeling: Dry all RT samples completely in speed vac. Resuspend each dried sample cDNA in 4.5 uL of 0.1M sodium carbonate buffer, pH 9.0. Mix up and down with pipette for good mixing. Dissolve and mix thoroughlythe Cy™ dye to be used with 20uL of DMSO. Tpically Cye dyes contain four applications in each vial. Mix 4.5 uL of dye with prepared cDNA sample. Incubate in the dark for 2 hour @ R.T. Mix RT-PCR products with 35uL 100mM NaOAc PH 5.2 and mix by vortexing. Mix with 250uL Qiagen Buffer PB and filter through spin columns (13,000 rpm). Wash spin column with 750 uL of Buffer PE (Qiagen). Spin an additional 1 min. with the same collection tube and discard the flow through. Elute labeled cDNA 2 times with 30 uL of EB Buffer (Qiagen). Combine different labeled probes Speed vacuum sample until dry (not too dry, i.e. don’t leave in speed vacuum for over 1 hour). Combine different labeled probes and speed vacuum sample until dry. Resuspend samples in 29uL sterilized dH2O
Harmen J. G. van de Werken1 and Marcel R. A. Verhaart
Growth protocol
same as channel 1
Extracted molecule
total RNA
Extraction protocol
same as channel 1
Label
Cy™5 dye (GE Healthcare)
Label protocol
same as channel 1
Hybridization protocol
Sample preparation steps for microarray hybridization: Prepare Prehyb buffer: 225mL water, 75mL 20X SSC, 0.3g SDS, 0.3g BSA. Preheat Prehyb buffer 45 min 42 degree C. Prehybidize slides at 42 degree C for 45 min. Wash slides by dipping 5 time in sterile dH2O at RT. Dip twice in isopropanol at RT. Dry slide in slide spinner. Add 1 ul COT1-DNA (20mg/ml) to samples. Heat probe mixture to 95oC in heat block for 2 min to denature DNA, do a quick spin. Add 30 ul of 2x hyb. buffer that has been preheated to 42oC to each tube. Hybridization buffer: 27.2uL water, 20uL 20X SSC, 8uL 50X Denhardts solution, 24uL formamide, 0.8uL 10% SDS. Apply hyb. mix to slide and cover with a 20x60 mm polyethylene hydrophobic coverslip. Place slide in water proof container, cover well with foil and place in 42 degree C bath for 18-20 hours. The following is a standard hybridization method that seems to work well for this procedure: 1 wash cycle at 42 degree C with 2X SSC w/ 0.2% SDS. 1 wash cycle at RT with 0.5X SSC w/ 0.2% SDS. 3 wash cycles at RT with 0.5X SSC
Scan protocol
Images were aquired with a scanarray 5000 (PE) with 2 lasers. Channels were balanced using the line scan feature and by balancing the overall signals without the use of external standards
Description
Scatterplots were used to verify overall quality of the data making sure both channels are equal.
Data processing
Signals for each channel were obtained using ScanArray express and were background substrated using the median local intensity around the spot. Once all the slides were quantitated, data from the loop was analyzed with JMP Genomics 3.0 (SAS, NC), using a mixed effects ANOVA model [Wolfinger et al. J Comput Biol 8 625-637 (2001).
