Differentiating xylem harvested from a Populus deltoides genotype D124 after six weeks of growth in a greenhouse.
Growth protocol
Plants were received as as hardwood cuttings. After rooting, bud break and shoot elongation, fresh softwood terminal cuttings were harvested and placed in rooting media pellets (Jiffy Forestry Products, Lorain, OH) for two weeks. Rooted cuttings were planted in two gallon pots and grown for six weeks in ebb-and-flow benches in a greenhouse under long day conditions (16h light:8hr dark) and standard nutrient regime (Hocking’s Modified Complete Fertilizer) supplemented with 25mM nitrogen (NH4NO3).
Extracted molecule
total RNA
Extraction protocol
At harvest, the main plant organs (stems, roots, leaves and sylleptic branches) were collected separately. Stems were further dissected into secondary xylem tissue and phloem/bark/immature xylem. Leaf, secondary xylem, and root tissue from two biological replicates of each genotype were selected for gene expression analysis. All tissue was flash-frozen in liquid nitrogen immediately after harvest and stored at -80° Celsius (C) prior to lyophilization and subsequent RNA isolation. RNA was extracted following the procedure described by Chang and colleagues (Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11: 117–121). After extraction, RNA samples were treated with RQ1 DNase (Promega USA, Madison, WI), purified in RNAeasy Plant Mini Kit columns (Qiagen USA, Valencia, CA), and integrity evaluated on 1% w/v agarose gels.
Label
Cy3
Label protocol
RNA was converted to double-stranded cDNA (SuperScript Double Strand cDNA Synthesis Kit, Invitrogen USA, Carlsbad, CA) with oligo-dT primer (Promega USA) according to the manufacturer’s protocol, except that synthesis of first and second strands were extended to 16h. Resultant ds-cDNA was labeled using cy3-tagged random 9mers and Klenow fragment for 2h at 37°C, prior to denaturation and hybridization to a microarray.
Hybridization protocol
Cy3-labeled samples (1-4 ug) were resuspended in hybridization buffer and hybridization component A (NimbleGen, Madison, WI), denatured at 95C for 5 minutes, loaded in the microarray chamber and allowed to hybridize at 42°C overnight (16-20h). Slides were then washed twice in Wash I (15 sec and 2 minutes), once in each of Wash II (1 min)and Wash III (15 sec) and dried.
Scan protocol
Slides were scanned with a GenePix 4000B Scanner. Initial pre-scannign was performed at PMT Gain = 500 and Power (%) = 100. After the pre-scan, the PMT was adjusted to avoid saturated spots.
Description
All procedures were carried out at Nimblegen facilities (Madison, Wisconsin).
Data processing
Microarray data was normalized using the quantile normalization procedure described by Bolstad and colleagues (Bolstad BM, Irizarry RA, Astrand M, Speed TP. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias.
Bioinformatics. 2003 Jan 22;19(2):185-93.).
