2. CENTRUROIDES VITTATUS
• Common name: Striped bark scorpion
• Geographic range: South-central United States and
northern Mexico
• Specifically south facing slopes
3. 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
4. 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
5. 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
6. 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
7. 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
8. 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
9. 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
10. 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
11. AGAROSE GEL OF AMPLIFIED CDNA
PRODUCT
1a 2
a
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
12. 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
13. 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
15. 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
16. 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 Bowman
Dr. T. Yamashita ATU Biological Sciences
Dr. Douglas Rhoads , UA-F Biological Sciences
Drs. TKS Kumar & Srinivas Jayanthi,
UA-F Chemistry & Biochemistry
17. 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)
South facing slopes encounter more sunlight in the northern hemisphere than north facing slopes
This venom is presumably produced in the gland housed within the telson.
Problems include extended depolarization to tail current decay to a mixture of both. Fewer channels opened/activation range = tail current decay
Quantifying different types of sodium toxins and seeing which sodium toxin is the most active amongst the species as a whole
Checking to see toxicity of scorpions over geographic range
iScript Rxn mix
iScript Res Trans
Nuclease free H2O
RNA Sample
After cDNA synthesis an agarose gel was made and cDNA was ran across the gel
