2. Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada
3. Metabolic Disorders and Complications Program, and Brain Repair and Integrative Neuroscience Program, Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada
4. Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada
5. Department of Genetics, Harvard Medical School, Boston MA, USA
1. Go to www.wormbase.org
2. Search for gene of interest (e.g. sod-2)
3. Click on “Location”. This will show you if there are multiple transcripts. You may want to target a specific transcript or try to design primers that target all of them.
4. Click on “Sequences”. Under “Coding Sequence” all of the transcripts for the gene of interest will be displayed.
5. Copy the spliced RNA + UTR sequence to microsoft word (in order to keep a record of where your primers are targeting).
6. If you are designing qPCR primers to measure RNAi knockdown, identify the region of the transcript where the RNAi clone is targeting. At least one primer should be outside of this region. We find that often primers targeting the untranslated regions (UTR) bind outside of the RNAi targeted region.
7. We use Primer 3 to pick primers (bioinfo.ut.ee/primer3-0.4.0/). If possible, it is best to pick at least one primer that overlaps spans two different exons. By designing a primer that contains parts of two exons, this primer will not be able to bind to DNA, which has introns present. We normally aim for the amplified region to be 100-200 bp. In the word file, we normally indicate the primer locations and note the expected size of the amplified region.
8. As a final check, we run the primer pair through an in silico PCR website: http://genome.ucsc.edu/cgi-bin/hgPcr. Be sure to select C. elegans. Because the primers are specific for spliced RNA, they should not amplify any DNA sequences.