QUANTIFYING SODIUM TOXIN GENE ACTIVITY IN THE SCORPION, CENTRUROIDES VITTATUS

Cody Chivers

CENTRUROIDES VITTATUS

• Common name: Striped bark scorpion• Geographic range: South-central United States

and northern Mexico • Specifically south facing slopes

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

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

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

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

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

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

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

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

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

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

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

RT-PCR GRAPH

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

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 

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)

QUESTIONS?