Augsburg College
RNA-Seq: Daphnia
Magna
Elissa Henderson
12-5-2015
Elissa Henderson
December 5th, 2014
RNA-
Seq:
Daphnia
Magna
Introduction
What is RNA-Seq?
RNA- Seq is a next-generation approach to producing many sequences from RNA
samples from model organisms, RNA-Seq is a useful took in measuring gene expression,
discovering complete transcripts and characterizing polyadenylation and alternative splicing.
The goal of RNA-Seq is to characterize the transcriptome of an organism, in which we selected
Daphnia Magna. RNA-Seq can reveal a “snapshot” of quantity of RNA from an organism’s
genome at a given moment in time. The steps of RNA-Seq are shown below.
Daphnia Magna as a model organism
Daphnia magna is a cladoceran fresh water flea, native to North America and Eurasia.
Daphnia are a commonly used model organism in biology labs. They are a good model
organism for biologist because they are easy to culture and require a simple environment to
maintain them. They also have a fast life cycle and mature very fast, so reproductive turnaround
is decently fast.
Overview
The first step to isolate the genes of interest, was to isolate the genomic DNA from our
model organism, Daphnia Magna, and extract its RNA. This was done by grinding
approximately ten Daphnia into a micro centrifuge tube, grinding the Daphnia up with a pestle,
and collecting the resulting supernatant. By doing this we disrupted the cells of the Daphnia.
Then, we used PCR to amplify the target genes. Two primers were used during PCR to target
regions of the genes before amplification. After isolating and amplifying the target genes, we
used gel electrophoresis to separate the PCR products and DNA fragments by size for analysis.
The final “in Lab” step was to perform a purification process to assure that DNA was purified
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December 5th, 2014
from the gel. Once the DNA is purified, we will collect our Daphnia DNA in to a clean centrifuge
tube we would theoretically have sent our finalized collection of cDNA fragments to be
sequenced in a different lab using chain-termination or short-read sequencing techniques. Due
to time, and financial reasons, we did not go on to send our samples in to be sequenced, and
instead will look at some already completed results on the model organism Arabidopsis.
The objectives of this project were to practice and perform basic molecular biology
techniques used for cloning and sequencing and to analyze our final samples and results using
gel electrophoresis and bioinformatics tools.
Methods and Results
I: Extract RNA from Daphnia Magna
For extracting the RNA of Daphnia we used a protocol written by Michelle Gradekman
from her Total RNA Miniprep Kit Instruction Manual. The steps can be seen below.
1. Put 10 adult Daphnia into a 1.7-mL RNase-free micro centrifuge tube. Remove APW.
2. Add 350 microliters of buffer into the tube
3. Drill/lyse the tube to grind up the Daphnia
4. Centrifuge 5 mins, and spin down insolubles.
5. Place supernatant in a new tube
6. Add equal volume of 70 percent ethanol to that of the supernatant, vortex, and
pipette 700 microliters of this solution into a RNA mini column. Centrifuge 1 min.
7. Add 500 microliters WF buffer and centrifuge+ discard flow through.
8. Add 700 microliters WS buffer and centrifuge+ discard flow through
9. Centrifuge and place column in clean 1.7-mL RNase-free tube.
10. Add 20 microliters of nuclease-free water to the column’s membrane and let stand
for one min.
11. Repeat step J.
12. Put the extracted RNA onto ice, and chill in -80 degree freezer for next step.
For this step we used a spin column to isolate the RNA, which worked by separating the
pellet and the supernatant. During the centrifuging, the RNA binds to the silica matrix of the
column because the water in it will bind with the properties of the silica, creating a negatively
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December 5th, 2014
charged O group that will bind to the chaotropic salt used to denature the proteins, and finally
binds to the phosphate group of the RNA. Solutions of the same ionic charge will remain bound.
For our protocol on RNA-Seq we utilized Dr. Arthur Hunt’s custom protocol for utilizing
alternative polyadenylation. Alternative polyadenylation is important for regulating gene
expression. This protocol is divided into three steps: RNA preparation, cDNA synthesis, and
PCR purification. We use the Poly (A) enrichment step because we are utilizing Eukaryotes for
our experiment.
II. Poly (A) Enrichment
Steps
1. Aliquot 10 microliters of the Oligo (dT) beads, and add them into the Daphnia RNA from
the RNA extraction described above.
2. Wash beads with 100 microliters of binding buffer, remove supernatant and use
magnetic strip to capture the beads.
3. Resuspend the beads in 50 microliters of binding buffer.
4. Bring RNA to 20 microliters with RNAse-free water.
5. Heat RNA to 65 degrees C, cool on ice, add to washed beads and incubate at room
temp for 5 minutes.
6. Collect beads again using magnetic stand, discard supernatant
7. Wash beads twice with 100 microliters of wash buffer, and collect beads with magnetic
stand again after each wash.
8. Remove supernatant and add 15 microliters of 10mM Tris-HCL and heat at 80 degrees
C for 3 minutes to elute the mRNA.
9. Removed beads with the magnet and place supernatant in new RNAse-free tube on ice.
III. RNA fragmentation, cDNA Synthesis + Cleanup
Steps
1. Mix 14.5 microliters of organism’s RNA, 1 microliters of Reverse transcriptase (RT)
primer, and 5 microliters of 5x 1st strand buffer
2. Heat to 95 degrees C for 2 mins, chill
3. While still cold, add 2.5 microliters of 10X dNTPs for reverse transcription
reaction, 1 microliters 100 mM DTT, .5 microliters RNase inhibitor, 1 microliters
SMARTSCRIBE.
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December 5th, 2014
4. Mix + incubate for 120 minutes at 42 degrees C.
5. Heat to 70 degtrees C for 5 minutes.
6. Add 20 microliters AMPure beads. Incubate for 8 minutes at room temp.
7. Separate beads once again using the magnet stand, remove and discard
supernatant.
8. Add 200 microliters of fresh 80 percent ethanol, mix and collect beads. Remove
and discard supernatant.
9. Air dry the washed beads at room temp for 5 minutes.
10. Add 25 microliters 10 mM Tris HCL. Mix and collect the beads with the magnetic
stand. Remove supernatant to new 500 microliters thin-walled micro centrifuge
tube.
11. Repeat steps 6-10.
IV. PCR Amplifications
The purpose of PCR is to amplify a segment of DNA or RNA, what is done is the sample
is heated so the cDNA can denature and separate into two pieces of single-stranded DNA. Then
we use an enzyme called Phire to bind the two new strands of DNA, using the original strands
as templates. The reason Phire is used is because it is a hot-start enzyme in that it can
withstand very high temperatures and still function properly, and high temperature is very
necessary for the initial denaturation of the DNA.
For our PCR reaction we will be using two primers: PE-PCR1 and PE-PCR2. The
annealing temperature is 60 degrees C for a time of 15 seconds, the extension time is for 60
seconds. We will run 15 cycles, so our library is not too large. The goal of this step is to smear
200-500 base pairs, and if successful tags can be purified and amplifications can be scaled up
using AMPure protocol.
V. Gel Electrophoresis
The PCR reaction from the previous step can now be analyzed by agarose gel
electrophoresis. For this, a 1% agarose gel with wells is cast using a well comb. WE then waited
for the gel to solidify in the well
Steps
1. Remove the comb, transport the gel holder to the gel box.
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December 5th, 2014
2. Pour 1X TAE buffer with the same concentration of EtBr added to it that the gel contains
(10 microliters per 100 mL of buffer). Submerge gel in buffer.
3. Pipette 10 microliters of the DNA ladder into the first well.
4. Pipette 25 microliters of sample into a different well, take note of which lane it’s loaded
in.
5. Put lid on the box, plug cords into voltage supply, with Neg charges at the top.
6. Set voltage to 100V and allow current to run for an hour.
7. Once Gel is done running we will drain the buffer off the sample, and go on to image the
gel using the BioRad Gel Documentation Station. Our gel Result can be seen below.
Lanes 1-3 were all filled with molecular weight/DNA ladder, the 4th lane is our sample.
The bands that traveled further towards the positive pole are smaller than the ones that
travel a shorter distance through the gel. The presence of more than 1 band from the same well
indicates that there are different sized molecules in that sample. Intensity of bands indicate the
amount of PCR product that is in the sample. So it is significant to get the most prominent band
in your purification.
After imaging the Gel it is important to cut the bands out for purification. We then cut out
the most prominent bands and placed them into a new 1.7ml tube.
VI. Purification of DNA from Agarose Gel
For this portion of the experiment we will use a protocol from the Epoch GenCatch
Advanced Gel Extraction Kit Instruction Manual written by Michelle Grafelman, and modified by
Matt Beckman.
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December 5th, 2014
Steps:
1. Measure the mass of the gel slice, ours was 200mg. Place it in a sterile 1.7-ml tube.
2. Add 3 volumes GEX Buffer to 1 volume of gel
3. Incubate tube at 55 degrees C for ten minutes, invert tube every 1-2 mins
4. Load 700 microliters of the dissolved gel/buffer mixture into a gel extraction column,
positioned in a round-bottom collection tube.
5. Centrifuge for 30 seconds at 5000rpm, discard flow through
6. Add 500 microliters of WN buffer to wash the column, centrifigue for 1 minute at
5000rpm, discard flow through.
7. Add 500 microliters of WS buffer to wash the column, centrifuge for 1 minute at
5000rpm, discard flow through
8. Centrifuge the column at 12,000rpm for three minutes to remove residual ethanol which
can affect the quality of DNA and inhibit future enzyme reactions, Place the column in a
new tube.
9. Add the 20 microliter Elution Buffer onto the center of the white membrane within the
tube and let the column stand for two minutes. Centrifuge for one minute at 12,000rpm.
10. Repeat step 9 but DO NOT discard the flow through, as this is now our DNA in the
elution. Store in -20 degrees c area.
Uncertainties
Our results could have been affected by factors as small as not wearing gloves or poor/inexact
measurements. Contamination is probable during PCR, when purity is exceptionally vital to a
successful product.
VII. Sequencing
The resulting sequence reads that we would have obtained are then aligned with the
reference transcriptome and go on to generate a base-resolution expression profile for each
individual gene. As I had mentioned in the overview, we were not able to send our particular
libraries out to be sequenced due to time constraints, we did however get to look at a previously
completed RNA-seq library pertaining to the model organism Arabidopsis. The Analysis of those
results can be seen below.
Analysis
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December 5th, 2014
The basis of RNA-Seq is to be able to ultimately see the varying levels of gene
expressed and what genes are or aren’t expressed at a specific moment in an organism’s
development. The library we attempted to analyze was a previously done sample uploaded to
the genomics education website named DNAsubway. DNAsubway has many RNA-seq data
sets pre-analyzed for our use. Some additional different that resulted from other’s RNA-Seq
results and data were given to us to analyze. These can be seen below.
The first sample is an example of a graph we can get as an end result of RNA-Seq. This
graph shows differential expression. A gene is considered differentially expressed if one can
observe that a difference in read counts between two conditions is much greater than one would
expect due to just random variance. This graph tells of the varying levels of expression of
particular genes over a certain time period. The graph below is from an unspecified organism
from its development from 0-14 days. The graph below shows a variety of genes along the right,
and whether they are up or down regulated in terms of expression. From looking at the below
figure we can see that the pattern of regulation is shown from green to red, or red to green. The
green to red would indicate that the gene is upregulated, meaning that they are genes that
regulate some aspect of a cell or system by upping/increasing the production of something. So
in the case of a red to green pattern, i.e.; the pluripotency genes, we see that expressivity
decreases as the days pass by. This would be something that is common in stem cell, so the
expressivity is high prior to cell differentiation.
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December 5th, 2014
The below graphs are also samples of what one can get from RNA-Seq. The graph on
the right represents the data from wild-type Arabidopsis, and the graph on the left represents
non-wild type Arabidopsis. The below graphs can give us an overview of the range of quality
values across all bases at individual positions. The key to reading the below graphs is as
follows:



The central red line is the median value
Yellow boxes = inter-quartile range (25-75%)
Upper and lower whiskers = 10% and 90% points

Blue line = mean quality
The X axis denotes the position in base pairs in the sequencing read. The y axis on the
below graphs represent the quality scores. A higher score will result in a better base cell.
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December 5th, 2014
Behind the boxes is a division on colors that divide the data into three call qualities. The
green is very good quality calls, orange is decent quality, and red represents bad quality
calls. Looking at these two graphs we can conclude that both of these organism’s had
quality scores of between 25 and 33, which can be concluded as decent quality. Near the
end of the reads quality scores decrease however the average quality score is still within the
green and orange range of the graph meaning that quality can be considered as good.
The next graph provided to us is called a scatter plot. It is one of the many types of
graphs that can be obtained by our RNA-seq experiments. It can be seen below. These plots
can help recognize any potential environmental changes that may be influencing genes
between two organisms of the same species, and trends in gene expression between them.
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December 5th, 2014
Conclusion
RNA-Seq is a newer technology for measuring gene expression that is quickly replacing
older techniques such as microarrays in labs around the world. During RNA-Seq, mRNA are
converted into cDNA that is used to generate a sequencing library. RNA-seq gives us the ability
to quantify and discover various RNA profiles, as well as find new genes for functional analysis.
RNA-Seq is still a new technique, but it still proved superior to past methods to measure gene
expression. It is also more cost effective which is beneficial as well.
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December 5th, 2014
References
1. Hunt, A. (2015). A Rapid, Simple, and Inexpensive Method for the Preparation of
Strand-Specific RNA-Seq Libraries. Polyadenylation in Plants, 1255.
2. RNA-seqlopedia. (n.d.). Retrieved December 11, 2015, from
http://rnaseq.uoregon.edu/
3. RNA-Seq Blog. (n.d.). Retrieved December 12, 2015, from http://www.rnaseqblog.com/
4. Understanding Analysis Outputs - Maverix. (n.d.). Retrieved December 12, 2015,
from http://www.maverixbio.com/outputs/