Rice Genetics IV - IRRI books - International Rice Research Institute

Table 1. Selected cDNA libraries from salt-stressed plants.Name Organism Tissue Stress condition a Plant ageHB H. vulgare (Tokak) Leaves Drought; 6 and 10 h ~3 wkHC H. vulgare (Tokak) Roots Drought; 6 and 10 h ~3 wkMC M. crystallinum Roots No stress 5–6 wkME M. crystallinum Roots 400; 6 h 5–6 wkMF M. crystallinum Roots 400; 12 h 5–6 wkMG M. crystallinum Roots 400; 30 h 5–6 wkMH M. crystallinum Roots 400; 78 h 5–6 wkMI M. crystallinum Seedlings 250; 3 d 14 dML M. crystallinum Flowers and seedpods 500; 6 wk >12 wkMM M. crystallinum Epidermis 500; 6 wk >12 wkMN M. crystallinum Side shoots 500; 3 d 6 wkMO M. crystallinum Meristems No stress 5 wkMP M. crystallinum Meristems 500; 3 d 6 wkOA O. sativa (Nipponbare) Roots 200; 19 h 3–4 wkOB O. sativa (Nipponbare) Leaves 200; 19 h 3–4 wkOC O. sativa (Pokkali) Roots No stress 1 wkOD O. sativa (Pokkali) Roots 150; 1 d 1 wkOE O. sativa (Pokkali) Roots 150; 2, 3 d 1 wkOF O. sativa (Pokkali) Roots 150; 1 wk 2 wkOG O. sativa (Pokkali) Leaves 150; 1, 2, 3 d, 1 wk 1 wkOH O. sativa (Pokkali) Leaves No stress 1–2 wkZA Z. mays (B73) Roots 150; 24 h 2 wkZB Z. mays (B73) Leaves and shoots 150; 24 h 2 wkaStress is expressed as mM NaCl and length of the treatment. NaCl is applied as a shock treatment.and filtered. PCR products were eluted with 10 mM Tris-EDTA. About 1.2–2.4 µg ofPCR products were dried and the pellets dissolved in 6 µL of 1X SSC for printing.Microarrays were produced by using the Omnigrid spotter (GeneMachines, San Carlos,Calif.). Slides, coated with either polylysine or aminoalkylsilane, contained cDNAsspotted in duplicate or triplicate. Only amplicons longer than 400 bp were printed.Preparation of labeled probesFluorescence-labeled probes were prepared from RNAs by incorporation of fluorescentnucleotide analogs during first-strand reverse transcription. Each reaction (50µL) consisted of 1 µg mRNA, 200 ng of in vitro transcripts as human control mixture,2 µg of oligo(T) primers, 0.5 mM each of dATP, dCTP, and dGTP, 0.2 mM dTTP, and0.5 unit of reverse transcriptase (Superscript II, GIBCO Inc.) in 1x reaction bufferand 2 nmol of either Cy3-dUTP or Cy5-dUTP (Amersham, Pharmacia). RNA andprimers were heated to 65 °C (10 min) and quenched on ice before adding the remainingreaction components. The RT reaction proceeded for 10 min at 42 °C preincubationfollowed by 90 min at 42 °C. Buffer exchange, purification, and concentration ofcDNA products were by microfiltration (Qiagen). Labeled targets were collected bycentrifugation after the addition of 0.1 volume of 3M potassium acetate and 1 volumeof isopropanol. The dried pellets were reconstituted in 20 µL of 5X SSC/0.1% SDS/50% formamide and denatured (95 °C) prior to use in hybridization.348 Bohnert et al