Katerina Holan
2015 SURF Final Report
Research Interests
My research interests are to improve or alter crop genetics through plant breeding or
biotechnology in hopes of creating more sustainable agriculture.
Analyzing RNA-seq Data to Determine Transcriptome Differences for NUE in Maize
Abstract
This research took samples from various maize lines and tissues that were grown in both
high and low nitrogen in order to determine differing gene expressions derived from those two
nitrogen levels. This involved extracting RNA from each sample and checking its quality with
both a formaldehyde gel and a concentration measurement. The samples were then purified with
a DNAse protocol. Each sample’s RNA concentration was then calculated again to ensure good
quality RNA. This fall, RNA-seq will be done on all of the samples to determine the expression
level of each gene product. These expressions can then be compared and analyzed to determine
if the differing nitrogen levels resulted in differential expression.
Introduction
My original research project for this summer involved working on a research project in
the Stephen Moose Corn Functional Genomics Lab at Illinois in the Edward R. Madigan
Laboratory. The overall research project is seeking to create a database called NutriNet, which
will show the relationship of genes and gene products, specifically RNAs, involved with nitrogen
use efficiency (NUE) in maize. This research is focusing on 30 different maize lines including
the Illinois High Protein (IHP) and the Illinois Low Protein (ILP) lines. My portion of this
research was to extract RNA from almost 300 samples of maize. The ear shoot and leaf samples
were taken from each line in the summer of 2014 prior to flowering. There were six samples for
each line from each nitrogen level; an ear shoot and a leaf sample were taken from three separate
plants.
Methods
This summer, most of my time was spent on perfecting and executing the RNA extraction
protocol. RNA is a particularly finicky molecule to work with as it is easily degraded. I had to
make sure that my protocol, reagents, and workspace were clean before starting each RNA prep.
When I first started, I went through a CTAB/acidified phenol RNA extraction on a few samples
and then ran the subsequent RNA products on a formaldehyde gel to ensure that my protocol was
working properly. Unfortunately, there were no RNA bands after running the gel when exposed
to UV light. This was troublesome, as it meant any number of things could have gone wrong. It
could have been RNA degrading enzymes (RNAses) in any of the reagents, on the pipettes or
tips I was using, or in the gel itself. At the very worst, it could have been that the RNA in the
samples that were collected last summer was already degraded. I ran the RNA extraction prep
on some teosinte (the ancestor of modern day maize) and spent the next couple of weeks
troubleshooting the gel prep with those teosinte products. After several gels and much
frustration, I finally saw bands on my gel. This was the best case scenario because it meant that
the protocol, reagents, and most importantly, the samples, were not the problem. I then spent the
next few weeks methodically grinding samples, extracting RNA, and running the subsequent
products on gels to check for bands (see fig. 1). I used the teosinte as positive controls to make
sure that my gels were running properly. As long as the teosinte band showed up, it meant that
my gel was running correctly.
Fig. 1: RNA gel from 5/4/2015. Lane 1 is a teosinte positive control.
Lanes 2-24 are experimental maize samples
RNA and DNA are both fairly similar molecules. To avoid extracting DNA along with
the RNA, a slightly different protocol is used, but often small amounts of DNA will come along
with the RNA anyway. In order to analyze RNA samples, the DNA must first be degraded.
After I finished extracting all of the RNA, I checked the concentration of the RNA samples using
an instrument called a Qubit. This machine will disregard any DNA left in the sample and only
display the concentration of RNA in that sample. With this information, I then used
corresponding amounts of reagents and ran a DNAsing prep on every sample. This DNAsing
prep degrades the DNA left after extracting the RNA, leaving clean RNA that can then be used
for analysis. After DNAsing all of the samples, I measured the concentration of the samples on
the Qubit once again to ensure that the RNA concentration remained steady. In most of my
samples, the concentration decreased due to what I believe was the RNA not precipitating out of
the DNAsing reagents. Even so, most samples have a high enough concentration on which to do
further analysis.
Conclusions and Future Research
I was unable to draw any conclusions as the samples still need to be sent off to be run
through a process called RNA-seq. This will determine the sequences of all of the different
RNA transcripts in each sample. Hopefully, there will be differential expression in some genes
for those plants grown in high nitrogen versus those grown in low nitrogen. The IHP and ILP
will be extremely useful for this research project. By comparing the differential expression
between these two lines and the rest of the experiment lines, hopefully I will be able to determine
which genes are important in nitrogen use efficiency. I am going to continue working on this
research this fall semester. Once I get the results back from the RNA-seq, I am planning on
using bioinformatics programs to edit, align, and organize the RNA-seq data. Then, I will be
able to find any differential expressions in these gene products based on the differing nitrogen
levels and maize lines.