genotype/variation: control cell line vector: GUS time point: 3 days of growth
Treatment protocol
The HP and the control cell lines of Populus nigra × maximowiczii used here have been described previously (Bhatnagar et al., 2001; 2002). The former expresses a mODC cDNA while the latter expresses the bacterial GUS gene (that serves as a control); both cell lines also express the neomycin phosphotransferase (NPTII) selectable marker. All transgenes are constitutively expressed under the control of a modified 35S CaMV promoter. This strategy has allowed us to treat both lines identically for maintenance of stocks during their culture history; e.g. growth in the presence of kanamycin. • Bhatnagar, P., Glasheen, B.M., Bains, S.K., Long, S.L., Minocha, R., Walter, C., and Minocha, S.C. (2001). Transgenic manipulation of the metabolism of polyamines in poplar cells. Plant Physiol. 125:2139-2153. • Bhatnagar, P., Minocha, R., and Minocha, S.C. (2002). Transgenic manipulation of the metabolism of polyamines in poplar cells: the catabolism of putrescine. Plant Physiol. 128:1455-1469.
Growth protocol
Liquid cultures of both the HP and control cell lines were maintained on a weekly subculture routine and harvested as described (Page et al., 2012). Over long term, solid cultures (callus) of the two lines were also maintained (on a monthly subculture routine) and used to restart the liquid cultures if they were lost due to contamination etc. All cultures, except the NT (non-transgenic) cells were grown in Murashige and Skoog (1962) medium (solid or liquid) containing 3% sucrose and 0.5 mg.L-1 2,4-D under 12 h light cycle (80-100 μE.m-2.sec-1 at 25±1 C. • Page, A.F., Minocha, R., and Minocha, S.C. (2012). Living with high putrescine: expression of ornithine and arginine biosynthetic pathway genes in high and low putrescine producing poplar cells Amino Acids 42:295-308.
Extracted molecule
total RNA
Extraction protocol
The cells from liquid cultures were collected by vacuum filtration on Miracloth, washed quickly with de-ionized water, and weighed. Total RNA was extracted as described by Page and Minocha (2004), and treated with the TURBO DNA-free kit (Ambion Inc, Austin, TX) to remove genomic DNA contamination. • Page, A.F., and Minocha, S.C. (2004). Analysis of gene expression in transgenic plants. In: Transgenic Plants: methods and Protocols--Pena, L., ed.: Humana Press. 291-312.
Label
Cy5
Label protocol
RNA (13.5 µg of each sample) was reverse transcribed using a SuperscriptTM Indirect cDNA Labeling System (Invitrogen, Carlsbad, CA), with oligo-dT primers. cDNA was purified using S.N.A.P. Columns (Invitrogen, Carlsbad, CA) and resuspended in 10 µl Coupling Buffer following the manufacturer’s instructions. Individual aliquots of Cy3 and Cy5 (RPN 5661, GE Healthcare, Piscataway, NJ) were resuspended in 5 µl DMSO, mixed with 5 µl of the cDNA in Coupling Buffer and incubated at room temperature for 1 h in the dark. The labeled cDNAs were purified using S.N.A.P. purification columns, and the yield and dye incorporation were evaluated using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE). Targets were prepared by combining 40 pmol of each labeled sample in an amber microfuge tube. This was flash frozen in liquid nitrogen and dried in a vacuum centrifuge before storage at -20°C.
Hybridization protocol
All microarray slides were checked using a dissecting microscope for uniformity of spots. Slides were prehybridized for 30 min on a slow rotary shaker in 5X SSC, 0.1% SDS, and 1% BSA, and rinsed 20 times in ddH2O. DNA was denatured by incubating slides in 95°C ddH2O for 1 min, followed by washing in 100% ice-cold ethanol for 15 s. Slides were dried by centrifugation (4°C, 2500 rpm, 2 min) and stored at 4°C. Hybridization was performed within 1 h of prehybridization. Targets were resuspended in 55 µl hybridization solution (50% formamide, 5X SSC, 0.1% SDS, and 0.1% BSA) and centrifuged at 14,000 × g for 1 min before denaturation at 45°C for 5-10 min. Hybridization was performed in Corning hybridization chambers (Corning Inc., Acton, MA) with Lifter slips (Erie Scientific Co., Portsmouth, NH) and incubated in a 43°C water bath in a hybridization oven for 36 h. Slides were rinsed in wash solution I (1X SSC, 0.2% SDS), prewarmed to 45°C, to remove the lifter slips. Slides were washed once in wash solution I for 15 min at 45°C with gentle agitation, once in wash solution II (0.1X SSC, 0.2% SDS) for 10 min, and twice in wash solution III (0.1X SSC) for 2 min each. Finally, slides were immersed in ddH2O for 10 seconds, 100% ethanol for 10 seconds, then dried by centrifugation (4°C, 2500 rpm, 2 min) and stored in darkness at room temperature.
Scan protocol
Slides were scanned using a VersArray ChipReader™ scanner (BioRad, Hercules, CA) at 5 µm resolution. Cy3 and Cy5 images were aligned and spots flagged using VersArray Analyzer 5.0 (BioRad, CA).
Description
391 cont Cy5 Mean (locBGC)
Data processing
For the analysis in the associated paper, local background subtraction and exclusion of flagged spots was performed. Signal intensity was then log(2) transformed and normalized by the LOWESS algorithm with a smoothing parameter of 0.2, using GeneGazer software (Bio-Rad, CA). Spots having intensities below 20 in both channels were excluded. local background subtraction for each sample and channel are included the sample data table.
