Biomedical Science Division

1. - + SMN U Full Length ∆7 ∆5 ∆57 Paraquat • George Washington Carver Internship Program 2011 • July 29th 2011 Biomedical Science Division Alexandra Myhal, Eric Ottesen, and Dr. Ravindra Singh Quantification of Human SMN Spliced Variants by qPCR - + - + - + - + Abstract Quantitative PCR (qPCR) is an accurate method of determination of relative abundance of transcripts expressed in normal and pathological conditions in a particular cell type. Here I used the StratageneMxPro 3005P qPCR machine from Agilent Technology to determine the level of several spliced isoforms of the survival motor neuron (SMN) gene. We particularly focused on inclusion and skipping of exon 7, which is associated with spinal muscular atrophy (SMA), a leading genetic disease of children and infants. The SMN gene is present in two copies, SMN1 and SMN2, which are differentially spliced. SMA occurs when SMN2 is the only gene present, as it is unable to compensate for the loss of SMN1 due to increased skipping of exon 7 (2). Our findings underscore that exon 5 and exon 7 are more susceptible to skipping under oxidative stress. Further study will be needed to determine the extent of skipping of different exons in different tissues. C Results A Figure C. A logarithmic graph of the relative abundance of isoforms in the different SHSY-5Y cell treatments. The absolute quantity of each isoform was determined against a serial dilution of plasmid DNA. The relative abundance was determined by taking the percentage of each isoform compared to the total expression of SMN (SMN Universal). Each bar represents the average of the three replicate reactions. The bar graph shows there is increased skipping of ∆7. ∆5, and ∆57 in the paraquat treated neuronal SHSY-5Y cells. Also there is a decrease in the FL isoform in the paraquat treated neuronal SHSY-5Y cells. Figure A. RT-PCR of neuronal SHSY-5Y cells from exon 4 to 8. This figure shows the specific isoforms we later quantified by qPCR. The relative intensity of each band was used as an estimate of isoform quantity. Our results confirm an inhibitory effect of paraquat on SMN splicing. We found a higher increase in co-skipping of exon 5 and 7 skipping of either exon 5 or 7. Suggesting a possible correlation between skipping of exons 5 and 7. These findings indicate that SMA patients exposed to paraquatwould have increased severity of disease. Discussion Materials and Methods B The neuronal SHSY-5Y cells were exposed to 1mM of paraquat for 24 hours which induced conditions of oxidative stress. RNA was isolated from paraquat and PBS treated cells by Trizol (Invitrogen) and the cDNA was generated using SuperScript (Invitrogen) III Reverse Transcriptase using the manufacturer’s protocol. The qPCR machine used for this research was the StratageneMxPro 3005P and the data was analyzed by MxPro QPCR Software version 4.10, both from Agilent Technology. The Master mix for each assay reaction used was the Brilliant II SYBR Green QPCR Master mix manufactured by Agilent Technologies. The primer sets for each assay were designed to anneal to specific exon-exon junctions to distinguish different spliced isoforms. Our quantitative assays showed increased skipping of exon 5 and exon 7 in the neuronal SHSY-5Y cells under conditions of oxidative stress. Conclusion References • Stratagene(2007). Introduction to Quantitative PCR. Methods and Applications Guide. • Monani, U. R., Lorson, C. L., Parsons, D. W., Prior, T. W., Androphy, E. J., Burghes, A. H. M., & McPherson, J. D. (1999). A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from copy gene SMN2. Human Molecular Genetics, Volume 8 (issue 7), 1177-1183. - + Paraquat Figure B. The Relative Quantity assay of different isoforms in neuronal SHSY-5Y cells. This chart shows the relative concentration of each isoform in the different treatments. Above each assay is the region of interest and the exon-exon junctions where the primers annealed. Acknowledgements: This work was supported by grants from the United States National Institute of Health. We would like to thank the George Washington Carver Internship Program for their funding and support.