For each TSS, we kept all smRNAs in a ?3 3 offset window. Since the smRNAs with a common 3 end binding position on the EST share the same predicted cleavage Wortmannin Sigma site we considered them as one group and categorized the targets according to the offset distribution of these smRNA groups. We cate gorized the targets as follows category I. the ESTs targeted by a unique smRNA group with a perfect offset, category II. the ESTs targeted by a majority of smRNAs with a perfect offset and category III. the remaining ESTs targeted by a minority of smRNAs with a perfect offset. Quantitative RT PCR RT PCR reactions were performed as previously described. In brief, first strand cDNA was synthesized using oligo primers and Super Script III reverse transcriptase. PCR reactions were performed on an AB 7900 HT Fast Real Time PCR System.
1. 0 uL of 1 10 diluted template cDNA was used in a 10 uL reac tion. The amplification program was 1 cycle of 15 at 95 C, 35 cycles 15 at 95 C, 30 at 60 C, 30 at 72 C, and then fol lowed by a thermal denaturing step. All primers pairs of the tested genes showed Inhibitors,Modulators,Libraries a similar amplification efficiency to the one used for the ACTIN gene which was used as reference. Relative transcript levels of biosynthesis were calculated with the Ct Inhibitors,Modulators,Libraries method. Northern blot analysis Total RNA was separated on a denaturing 15% polyacryamide gel containing 7 M urea at 120 V for 2 hr. RNA was electrophoretically transferred to Zeta probe GT membranes at 40 V for 90 min and fixed by UV crosslinking. Membranes were incubated in hybridization buffer for 4 h at 42 C and then incu bated in the presence of 32P end labeled oligonucleotide probes at 42 C overnight.
Membranes were washed in at 42 C and radioactivity was detected Inhibitors,Modulators,Libraries using a Phosphorimager. RLM 5 RACE RNA ligase mediated 5 rapid amplification of cDNA ends was performed using the GeneRacer kit. The manufacturers protocol for 5end analysis was followed with the exception of the 5 de capping step. In brief, total RNA was isolated from whole caryopsis tissues at 6 10 DPA and ligated to a 5end RNA adaptor before being re verse transcribed using an oligo primer. Background The plant kingdom is usually associated with autotrophy as most plants produce their own nutrients Inhibitors,Modulators,Libraries via photo synthesis. Plant parasitism presents a divergence from this generalization as parasitic plants derive all or part of their nutrients and water from their host plants.
About 4,000 parasitic plant species are widely distributed among various taxa and over diverse environments, Inhibitors,Modulators,Libraries ran ging from arctic to tropical climates. Some of the best known parasitic plants include the Christmas ornament mistletoe, Tubacin the world largest blooming flower Rafflesia, the fragant oil producing sandalwood, and the debilitat ing agricultural weeds dodder, witchweed and broomrape.