Figure 5

RT-PCR analysis of RNA templates isolated by the Sucrose Prep. (A) Amplification of a specific SOC1 cDNA product from leaf tissue. Leaf material was harvested and frozen in liquid nitrogen then ground in Sucrose Solution and subjected to heating at 99°C for 1, 3 or 5 min. A control PCR was performed with DNA isolated according to Edwards et al. [9]. A second control was performed with cDNA made from fresh leaf material prepared from RNA isolated by extraction using a commercial RNA isolation kit (Qiagen). (B) Isolation of RNA was performed as in (A) and heat treated at 99°C for 5 min. The samples were subjected to RT-PCR analysis using AGC1-10 specific primers, which flank the two introns depicted in the schematic diagram (expected sizes, genomic: 1300 bp, Intron I: 520 bp; Intron II: 215 bp). The cDNA product from the mature mRNA (600 bp) was detectable in shoot meristem tissues (s-m) and weakly in floral tissue, whereas only the first splicing product was observed in leaf tissues due to variations in the RNA yield (i.e., indicated by the amount of DNA product in the reactions).