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Research Projects
My major projects to date have been: Lion Bioscience amplification,
Real Time-PCR and Linear RNA amplification. I have also assisted
in various animal studies.
Lion Bioscience
Clone Amplifications
My main project this term has been to add on 7008 genes to our
existing mouse clone set with the help of graduate student,
Josh Kwekel and undergraduate student Nakaiya Okan-Mensah. The
clones are amplified using PCR and their quality is determined
through gel electrophoresis. The PCR products are purified in
Millipore plates and resuspended in spotting solution. Every
5th plate is analyzed by gel electrophoresis and completed plates
are condensed into 384-well plates for printing.
Real Time PCR
This project is supervised by graduate student, Darrell
Boverhof. We are assessing induction of classical genes
from rat uterus samples that have been treated with various
estrogenic compounds using quantitative Real Time PCR (QRT-PCR).
During QRT-PCR a fluorescent dye (Sybr Green) binds to double
stranded DNA. Thus, as PCR product increases, the fluorescent
signal increases. By comparing this signal at the exponential
phase of amplification of unknown samples with standards, an
estimate on the starting amount of product can be made.
Linear Amplification
For many tissues, extracting sufficient RNA to perform microarray
experiments can be difficult. To overcome this, attempts have
been made to linearly amplify RNA such that there are no biases
in the genes of the amplified RNA. Under the supervision of
Dr. Jeremy Burt, we’ve developed a protocol to achieve
100 fold RNA amplification. To verify the linearity of amplified
RNA we have used microarrays and Real Time PCR. However, the
experiments done so far have not produced reliable linear results.
Animal Studies
I’ve assisted Darrell
Boverhof and Josh Kwekel
in a pilot metabonomics study of rats treated with ethynl estardiaol.
Also, I’ve gained experience in handling and harvesting
tissues of rodents (ovariectomized mice and rats) during an
ethynl estardiol time course and a TCDD dose response study.
Second Term
During my second term at the Zacharewski lab, my main projects involve the
evaluation of global gene expression changes due to estrogenic compounds and
mixtures under varying doses and time points in both in vitro and in vivo
models. The main steps for these studies are: treatments, tissue/cell
harvest, RNA extraction and quantitation, and microarray analysis. A reference
design is used for the array models - treated and untreated samples are labeled
using a dye-swap with time matched vehicle control samples and comparative
analysis is performed on a custom cDNA microarray platform. Selected active
genes from the subsequent analysis are verified by real time PCR analysis.
More specifically, my main project entails the evaluation of estrogenic
compounds, 17-alpha ethynyl estradiol (EE, an orally active estrogen) and
genistein (a phytoestrogen found in soy products) in an individual and mixture
dose responsive study. Both in vitro and in vivo model systems will be
examined to evaluate the synergistic, additive or competitive effects of the
mixture upon global gene expression. We are using mouse hepatocyte Hepa1c1c7
cells for the in-vitro model and liver tissue extracts from immature,
ovariectomized C57BL/6 mice for the in-vivo model.
Additionally, I will look at the translation-independent changes in gene
expression resulting from estrogen treatmen.in the in vitro model. Hepa1c1c7
cells will be treated with and without cyclohexamide (a translation inhibitor)
prior to treatment with 17B-estradiol (E2) and temporal changes in gene
expression will again be evaluated by microarray analysis to distinguish
primary from secondary responses to E2 treatment.
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