Submitted to: Maize Genetics Cooperation Newsletter
Publication Type: Research notes
Publication Acceptance Date: 1/31/2003
Publication Date: 7/18/2003
Citation: BLAKEY, C.A., HOUGHTELING, B., GOLDMAN, S.L., DEWALD, C.L., SOKOLOV, V.A., HAMMERSMITH, R. ISOLATION OF STAGE- AND PLOIDY-SPECIFIC FLORAL RNA IN TRIPSACUM DACTYLOIDES FOR CDNA LIBRARY CONSTRUCTION - A PILOT STUDY FOR LARGE-SCALE ISOLATIONS. MAIZE GENETICS COOPERATION NEWS LETTER. 2003. V. 77. p. 56-57. Interpretive Summary:
Technical Abstract: A pilot study was conducted to determine the physical stages of floral development for the isolation of pre-emergent ovules of greenhouse plants for eastern gamagrass accessions WW1582 (sexual) and WW1008 (asexual, apomic) in the range of 0.5 to 3.0 mm, and to develop the step-by-step procedure for harvesting tissues containing ovules with minimal contamination for high quality RNA isolations. The RNA isolations would be used in the creation of stage- and ploidy-specific cDNA libraries. Harvested pre-emergent spikes were sized and ovules were excised to determine an approximate physical correlation between fruitcase width and ovule width within the fruitcase. Based on these findings a set of pre-emergent spikes with fruitcases were divided into three classes, <0.5 mm (E), 1.0-3.0 mm (M), and > 3.5 mm (L) (all fruitcases used were less than 5 mm in width). Total RNA was isolated from these sample classes using a Trizol™ RNA isolation protocol (as provided by Sigma). Stage-specific cDNAs were generated using the SMART™ PCR cDNA Synthesis Kit (CLONTECH Cat# K1052-1). Small quantities of cDNAs were detected for each class, but in insufficient quantities for library construction. In order to enhance total RNA isolations, larger quantities of staged tissues of these accessions would be required. It was determined that the fruitcase width measurements would be altered to compensate for the higher quantities of advanced maturity sexual spikes relative to the availability of early apomictic spikes during the limited RNA isolation experiment.