Most of 18s transcripts began to amplify around cycle 15 where as the agrpmrna did not even begin to amplify until about cycle 35
Since ribosomes are so necessary in all cells, the gene must be highly expressed and up regulated. This control proves that the brains were dissected, rna extracted, and cunverted to cDNA successfully because of the amplification of the 18s gene expression.
If it was possible to specifically dissect the hypothalamus from the brain, AgRP might be able to be examined and analyzed more successfully. This could provide more conclusive evidence to determine the role of AgRP in regulating body weight.
Unfortunately, there still was not enough AgRP amplification to draw any conclusions on the role of AgRP on regulating body weight. There was some amplification, but those that did amplify were from both Tank one and Tank two so it was challenging to draw any conclusions. For further, if I could find a positive control with a piece of tissue that is known to have high levels of AgRP mRNA expression, I could test the custom assay to make sure it was designed properly. The hypothalamus is known to have the highest levels of AgRP of the organs.
Sigma Xi 2013 Kelly Giffey
The Correlation Between Agouti Related Protein Expression and Dietary Intake within Zebrafish, Danio rerios March 8, 2013 Kelly Giffey W.F. West High School
Relevance• Obesity Epidemic in the United States – Two/thirds population overweight• 8.3% of population has diabetes – 25.8 million people in the US • Leading cause of death – More than smoking • Leads to complications: – Heart disease – Stroke – High blood pressure – Kidney Disease – Nervous system disease
Background - Agouti-Related Protein• AgRP gene found in humans and vertebrates. – Most production hypothalamus (brain) – Regulates eating response • Signals for hunger
Why We Feel Hunger?stomach Ghrelin AgRP Neuron Melanocortin Hungry! HUNGRY! 3 or 4 Receptor
Why We Feel Satisfied?Food Eaten Leptin Secreted from fat tissue AgRP Neuron STOPFULL! MC3R/MC4R
Effects of AgRP Overexpression• High AgRP levels – Leads to hyperphagia • Excessive overeating – Overeating overtime causes obesity • Leads to other health complications• Obese people and mice have higher levels of AgRP (Pan, 2005)• Mice fed 20% less protein diet have higher AgRP expression (Pillot, 2011)• After ghrelin injection to rats, AgRP mRNA levels rose (Kamegai, 2001)
Zebrafish Model Organism for AgRP Gene Expression• Danio rerios – contain similar organs as humans• AgRP protein synthesis occurs in hypothalamus
Purpose and Hypothesis-To determine whether AgRP expressionincreases in fish experiencing inducedstarvation.-Starved Zebrafish would have elevatedAgRP mRNA expression in the braincompared normally fed fish.
Research Design Overview• Two identical aquariums – Control Tank: 15 fish fed normal diet. – Experimental Tank: 15 fish fed with induced starvation.• Manipulated Variable: Diet• Responding Variable: AgRP expression levels as measured by qPCR.
Research Design• The Study - over 9 days• Three 3 day intervals – 5 fish taken from each tank at end of intervals – Fish were to be fed • Experimental - 2% of mass (tank 1) • Control - 4% of mass (tank 2) – Average fish mass 500 mg in both tanks
Fish Euthanization• End of feeding cycles, 5 fish removed from both tanks – Placed into 2 separate beakers of water
Procedure Overview Tissue Dissection RNA ExtractionmRNA converted to cDNA (Reverse Transcription) cDNA used in RT-PCR
Results18S Control Amp 18S Control 18S Control AgRP AgRP Amplified Product AgRP Gene Expression
What do my Findings Indicate?• RNA extraction and conversion to cDNA was successful• Overall, AgRP expression in brain tissue low• 18S highly expressed in brain
Conclusions• It was originally hypothesized that the starved fish would have higher mRNA levels for AgRP.• At this time results do not directly support the hypothesis• However, additional research focus could eventually show that AgRP expression levels are directly related to food intake
Further Research• If hypothalamus possible to dissect – AgRP could closely be examined – Evidence could result in determining role AgRP on regulating body weight• 1st run, AgRP didn’t begin amplification until cycle 30 – Very low level of transcript – Repeated gene expression experiment with higher DNA concentration.
Further Research• Even with a higher concentration it still yielded the same results.• More severely starve the fish – Fish closer extreme starvation levels• Time period increase• Tissue Cell culture
Acknowledgements• My Parents• My advisors Henri Weeks and Mike Stratton• David Cogan and Trish from Applied Biosystems
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