aas genetics research
TRANSCRIPT
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QUANTIFYING SODIUM TOXIN GENE ACTIVITY IN THE SCORPION, CENTRUROIDES VITTATUS
Cody Chivers
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CENTRUROIDES VITTATUS
• Common name: Striped bark scorpion• Geographic range: South-central United States
and northern Mexico • Specifically south facing slopes
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PRODUCTION OF VENOM
• Scorpions release venom when fending off predators or when they are feeding• The venom is presumably made by the gland
housed within the telson• Much of this venom is composed of neurotoxins• These neurotoxins are released when the
aculeus of the telson has penetrated its opponent
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SODIUM TOXIN
• We are especially interested in the sodium toxins • Sodium toxins alter the kinetics of sodium
channel gating• Problems associated with toxin:• Extended depolarization• Tail current decay
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OBJECTIVES
• To determine the quantity of neurotoxin being produced• Compared with other active genes in the telson gland• Compared to body tissues from Centruroides vittatus
• To show support of venom production occurring within the telson gland
• To do this we have to go through various procedures
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TOTAL RNA EXTRACTION
• We performed total RNA extractions on 4 samples• 2 female body samples • 2 female telson gland samples
• Using the Aurum™ Total RNA Mini Kit cat #732-6820• Kit provided various solutions and equipment to
perform the total RNA extraction
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NANODROP
• We used a NanoDrop to determine the concentration of RNA in ng/μl• TF21 – 25.9• BF21 – 42.1• TF22 – 28.7• BF22 – 51.6• Samples were then placed in our Eppendorf
thermocycler for cDNA synthesis
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CDNA SYNTHESIS
• We mixed our RNA samples with a master mix that we made in lab• We used a specific thermal cycle for cDNA
synthesis• 25°C for 5 minutes• 42°C for 30 minutes• 85°C for 5 minutes
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CDNA PCR AMPLIFICATION
• In a similar fashion as cDNA production • A master mix was created in lab• Samples were placed into the thermal cycler
for ~2 hours• Two sample DNAs were added • 828G• 678
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CDNA AMPLIFICATION CONT.
• We added 2 different primers to each of our samples: Na 2/4 and Lco/Nan• 2 female body samples • 2 female telson gland samples• 828G genome• 678 genome• In total we amplified the cDNA of 12 samples
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AGAROSE GEL OF AMPLIFIED CDNA PRODUCT
1a 2a
5a4a3a 6a L 1b 2b 3b 4b 5b• 1 – F body tissue 21
cDNA• 2 – F telson tissue 21
cDNA• 3 – F body tissue 22
cDNA• 4 – F telson tissue 22
cDNA• 5 – 828G genome• 6 – 678 genome
• a – Na 2/4 primer• b – Lco/Nan primer
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REAL-TIME PCR
• Over the last 10 years this method has exploded in popularity• 25,000 publications made• With RT-PCR we are able to compare phenotypic
observations with quantitative, molecular data• We use this method to show how much mRNA is
being produced
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REAL-TIME PCR CONT.
• Conventional PCR products (amplicons) are detected via agarose gels• With RT-PCR the amplicon can be detected
as well as quantified in real time• This can be achieved by using fluorescently
labeled sequence specific primers• The measured fluorescence is directly
related to the amount of amplicon
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RT-PCR GRAPH
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FUTURE ENDEAVORS
• Quantify different types of sodium toxins to see which sodium toxin is the most active amongst the species as a whole• Checking to see how toxicity of scorpions over
geographic range varies
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ACKNOWLEDGEMENTS
This project was supported by the Arkansas INBRE program, with a grant the National Institute of
General Medical Sciences, (NIGMS), P20 GM103429 from the National Institutes of Health
Arkansas Tech University
Aimee BowmanDr. T. Yamashita ATU Biological Sciences
Dr. Douglas Rhoads , UA-F Biological Sciences Drs. TKS Kumar & Srinivas Jayanthi,
UA-F Chemistry & Biochemistry
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REFERENCES
• Taylor, Sean, Michael Wakem, Greg Dijkman, Marwan Alsarraj, and Marie Nguyen. "A Practical Approach to RT-qPCR—Publishing Data That Conform to the MIQE Guidelines." Methods 50.4 (2010): n. pag. Print.
• Huang, Qiuying, Linlin Zheng, Yumei Zhu, Jiafeng Zhang, Huixin Wen, Jianwei Huang, Jianjun Niu, Xilin Zhao, and Qingge Li. "Multicolor Combinatorial Probe Coding for Real-Time PCR." PLoS ONE 6.1 (2011): n. pag. Web.
• Rozen S and Skaletsky HJ, Primer3 on the WWW for general users and for biologist programmers, pp 365–386 in Bioinformatics Methods and Protocols: Methods in Molecular Biology (Krawetz S and Misener S, eds), Humana Press, Totowa, NJ (2000)
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QUESTIONS?