To begin, I checked the integrity of the RNA that I was using in the generacer kit. Everything checked out great and the RNA was of sufficient quantity and quality to use. I began the generacer kit using root RNA and the control HeLa RNA provided in the kit. Following each step in the protocol, I analyzed the integrity of the RNA via agarose gel electrophoresis. This allowed a confirmation that the product was maintained throughout the reaction. I finished the generacer kit on 1-28-11. On 1-31-11, I amplified the ends of the cDNA that was generated following the exact specifications provided in the protocol for the generacer kit. I also included all of the negative controls that were recommended in the kit. The first attempt at visualization via electrophoresis was not successful. A few bands were showing but they were very faint. Next, I concentrated the PCR products that were remaining (40 uL) and reran the gels. The HeLa control reactions were successful but there were no bands showing for either the 5' or 3' end of any of the WRKY genes.
In an attempt to determine why the bands were not showing up for any of the target genes, I performed a one step RT PCR reaction with both root tip and root RNA. This would determine if the three genes were actually expressed in the target tissues. The resulting bands were very faint and led to the conclusion that the genes were not highly expressed in root tips or root tissues.
To circumvent this problem of expression level in these tissues, I will extract RNA from whole seedlings that were allowed to germinate for 5 days. I will then perform the generacer protocol with this RNA. Additionally, I developed 2 new primers for both the 5' end and the 3' end of each gene, which have different binding sites and may give more specific results. I will use all three primers that I have for each gene when I analyze the products generated from the generacer kit.
Hopefully, with these changes I will be able to sequence the 5' and 3' ends of these genes. I will start next week by extracting the RNA and then proceed with the generacer kit.
