S.J. Kanchanavaleerat 5/7/2015
RNA Extraction – Evaluating the Quality of Tissue
Samples
Abstract
Tissue samples can be evaluated by analyzing their RNA. To obtain RNA, a process known as
RNA extraction is done to evaluate tissue samples. RNA extraction is an extensive process that
first involves setting up solvents for the RNA. After preparing the solvent, the solvent is used for
the RNA Purification process, which isolates the RNA so that the quality of the sample can be
evaluated. The purified RNA is then run through a gel and by the use of a bioanalyzer to assess
the integrity of the sample extracted.
Introduction
RNA extraction is the first step of analyzing tissue samples by separating different components
of cells to evaluate expression of specific genes that we are looking for. Hence the name, RNA
extraction seeks to find specific messenger ribonucleotides within the tissue for analysis. To be
able to analyze the specific ribonucleotides, we use polymerase chain reactions (PCR) to
duplicate the genes to create thousands of copies so that we have the RNA sample in a higher
concentration for better analysis.
When running an RNA Extraction we do not know the concentration of RNA within the tissues
we are evaluating. Following the written protocol for RNA extraction approximate will help
approximate how much RNA content we have in our sample after it is purified. RNA purification
aims to give us pure RNA content without any other nucleotides, but unfortunately if too much
solvent or wash is applied to the RNA we will get futile results because our RNA will become
diluted. For this reason the amounts of solvents applied to the RNA when it is isolated must be
done with as little solvent as needed to produce the greatest concentration of RNA possible. If
the concentration of RNA is not enough, it is very difficult to differentiate between specific genes
when the RNA gel is run to identify genes.
Our aim in this experiment is to generate the high quality RNA samples. The purpose of an RNA
extraction is to prepare genes with a high level of integrity, which means of high quality so that
we can depict specific genes that are affected on the tissue samples. When the integrity of the
genes is high, we will be able to easily interpret the genetic material extracted from the RNA
extraction process.
By properly executing an RNA extraction, we will be able to assess the sample by running the
sample through a RNA gel via electrophoresis and spectrometer. If the RNA samples are well
purified, then we will receive high quality data that is easily interpretable. We can identify
individual genes that are collected from the sample.
Methods
Buffer Preparation
Buffer must be prepared to create an environment where RNA can be extracted from tissue
samples. The buffer is prepared by adding 10 mL and 40 mL of ethanol (95-100%) to the 40 mL
and 160 mL Direct-zol RNA PreWash concentrate.
An RNA Wash Buffer must also be prepared in order to purify the RNA sample extracted. This is
prepared by adding 48 mL of 100% ethanol (52 mL 95% ethanol) to 12 mL RNA Wash Buffer
S.J. Kanchanavaleerat 5/7/2015
concentrate or 192 mL of 100% ethanol (208 mL 95% ethanol) to 48 mL RNA Wash Buffer
concentrate.
Sample Preparation
The samples of RNA must be prepared by setting up cell suspensions. The cell suspensions are
prepared by pelleting cells by centrifugation. Then a supernatant will form, and the cell must be
lysed directly in TRI Reagent. Doing so will create a separated layer on the top of the solution,
which is the RNA sample that we desire to analyze. Taking this top layer we isolate it into a tube
to centrifuge and lyse multiple times prior to RNA Purification. We let these tubes sit to create
an environment suited for RNA purification by allowing time for incubation, and then transferring
supernatants into RNase-free tubes.
Biological Liquids
The samples must be prepared as liquids before being RNA purified. To do this, three volumes
of TRI Reagent are added to each volume of liquid sample. Then the mixture is emulsified by
vortex and incubating for five minutes at room temperature. Then after, contents must be
centrifuged at 12,00 x g for 1 minute and then the supernatant must be transferred into an
RNase-free tube.
Tissue Homogenization
The RNA samples that are going to be analyzed need to be homogenized before putting them
into the prepared solutions. This is done by combining 500 ug of TRI Reagent with 50 mg tissue
in a homogenizer. Then after the homogenized sample is centrifuged resulting with particles
forming on the surface. To remove these particles, the homogenate must be centrifuged at
12,000 x g for 1 minute. Then the supernatant must be carefully transferred into an RNase-free
tube.
RNA Purification
With the prepared homogenate and buffers, the RNA samples are purified using a collection
tube to separate out other nucleotides and excess that will not be analyzed. The solvent is first
prepared by adding one volume of ethanol directly to one volume sample of homogenate
estimating a 1:1 ratio between the contents. This will be mixed with TRI Reagent by vortex.
The first major step to RNA Purification involves spinning the tubes to separate out unwanted
liquid content. This is done with a spin column with the sample inside a collection tube. The
collection tubes will be centrifuged for 1 minute, and excess liquid will run through to the bottom
of the tube. This excess liquid must be discarded.
Then after the solvent has run through the RNA sample, the first PreWash step must occur in
order to rinse the RNA prior to extraction. In this process, 400 ug of Direct-zol RNA PreWash tat
was previously prepared is added to the column and centrifuged for 1 minute. The flow-through
is then disgarded. This step is repeated one time.
After rinsing the RNA sample, the RNA must be washed to isolate the RNA from other contents
within the sample. To do so, 500 uL of RNA Wash Buffer from previous preparation are added
to the column and centrifuged for 1 minute. The flow-through is then thrown away. Although we
must ensure that the wash buffer is completely removed, and therefore the centrifuge will be
done for 2 minutes.
Lastly, DNA must be broken down to give us a pure RNA sample. To do this, 50 uL of DNase is
put directly in the column matrix and centrifuged for 1 minute.
S.J. Kanchanavaleerat 5/7/2015
RNA Sample Analysis
After extraction, RNA concentration must be evaluated using nano drops. Nano drops are based
off nucleic acid absorbance and concentration, which will reflect concentration of the RNA
collected after purification.
Nano drops are used in RNA Analysis to assess the content of the RNA extracted. Using a
spectrometer, a nano drop portion of an RNA sample is put onto the lens of a spectrometer to
read the concentration by putting intense light on our sample. Putting intense light on the
sample will produce an absorbance value and ratios so that we can interpret the content purity
of the RNA sample.
The purpose of this experiment is to extract high quality RNA samples. In order to check the
RNA quality, an RNA gel must be run using a special microgel dye based system known as
bioanalyzer. The intensity of the color will give the quality of the RNA purified after the
purification process.
The bioanalyzer is read by a systematic system called RNA Integrity Number (RIN). The RIN is
a standardized way to determine the quality of RNA samples acquired. Using the
electrophoresis done for the RNA, a florescent light is used to evaluate the integrity of our RNA
sample using ribosomal ratio. The graph of a bioanalysis will show peaks portraying intensity of
these ratios, showing us that we have a quality sample of RNA. The scale is evaluated from 110, 1 being degraded RNA and 10 being an exceptionally high quality sample of a certain gene.
Using the RIN system we can evaluate RNA samples in a standardized way so that we have
confidence in our sample of RNA after purification.
Results
In our methods we described two different methods of evaluating the RNA extracted from our
tissue samples. The first method is by using a nano drop to assess the protein and molecular
make-up of our data using absorbance and concentration. The second method of analyzing our
tissue is using gel electrophoresis, which helps us to identify which RNA messenger genes have
been extracted by band indications from reading the distance traveled by the gene from the
source that it was displaced in.
Interpreting RNA Content using Spectrophotometry
The first method of RNA extraction analysis is using a nanodrop. Using a nanodrop, we use a
device called a spectrometer to analyze our RNA sample by measuring absorbance values
using light intensity onto our sample. As little as 2 ng is needed for spectrophotometric analysis.
The 2 ng sample is placed in concentration onto a small lens that is put into a spectrometer, and
will give us graphic data to show the content inside our RNA.
In order to interpret our absorbance values, we must first identify the specific peak absorbance
that we desire from our data. About 260 nm is the peak absorbance of nucleic acids, and
knowing this we can use Beer-Lambert’s law [A = ᵋcl] to determine the concentration of our data.
Using 260 nm as our reference point to evaluate molecular content in our data, absorbance
ratios must be used to assess how pure the ribonucleic acids are in the sample. Generally,
proteins and phenolic compounds reflect 280 nm as their absorbance value and other organic
S.J. Kanchanavaleerat 5/7/2015
compounds reflect 230 nm as an absorbance value. Using these numbers, we develop two
ratios to help us determine the purity of the nucleic acid content.
The first ratio is a 260/280 ratio. This is the ratio between nucleic acid and other proteins or
phenolic compounds. This evaluates the amount of impurity referring to the protein content
within the RNA sample. A respectable ratio is between 1.8 and 2.1. If the ratio is lower than 1.8,
then this indicates that our sample is contaminated by protein.
The second ratio is a 260/230 ratio. It isn’t as prevalent as the 260/280, but is also important.
230 nm reflects organic compounds in general, which includes phenol, TriZol, and peptide
bonds. This ratio is relevant because it can indicate that our isolating solvents may still be
contaminating our data, meaning that we need to wash out the solvent to better purify the
sample. Generally above a 1.5 is acceptable for the ratio for the 260/230 ratio.
Below is a set of 30 samples of tissue that went through the RNA Extraction process. After
going through a spectrometer, concentration values were collected as well as the 260/280 and
260/230 ratios.
Animal
ID
883 002
905 020
905 050
888 040
891 050
889 002
909 030
864 020
906 003
891 001
888 002
906 001
882 013
880 022
883 050
908 022
889 003
888 003
890 001
882 002
909 040
879 012
898 010
902 010
888 020
906 010
898 030
Extraction
Tube
Conce
date
number
1912.02
10/15/2014
585.87
10/15/2014
667.53
10/15/2014
2840.22
10/15/2014
2699.46
10/15/2014
541.5
10/15/2014
230.463
10/15/2014
2974.83
10/15/2014
312.6
10/15/2014
1503.09
10/15/2014
4192
10/15/2014
117.72
10/15/2014
3255
10/15/2014
1863.66
10/15/2014
603.42
10/15/2014
134.31
10/15/2014
2004.12
10/15/2014
399.45
10/15/2014
174.11
10/15/2014
589.29
10/16/2014
3607.56
10/16/2014
982.8
10/16/2014
301.86
10/16/2014
710.67
10/16/2014
2020.68
10/16/2014
122.391
10/16/2014
653.34
10/16/2014
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
1
2
3
4
5
6
7
8
260/230
260/280
Gel result
1.13
1.3 repeat
1.41
1.68 good
1.67
1.75 good
1.5
1.4 repeat
2.16
1.79 repeat
2.14
1.73 repeat
1.37
1.75 good
2.2
1.77 repeat
1.29
1.7 good
1.69
1.55 repeat
2.15
1.77 repeat
1.35
2.02 repeat
2.21
1.8 repeat
2.17
1.8 repeat
2.04
1.81 good
0.86
1.43 repeat
2.18
1.81 repeat
1.44
1.8 good
1.28
1.44 good
1.55
1.71 good
2.18
1.78 repeat
2.19
1.82 good
1.29
1.7 good
1.56
1.74 good
1.83
1.8 repeat
0.71
1.72 good
1.69
1.78 good
S.J. Kanchanavaleerat 5/7/2015
878 010 2015.31
10/16/2014
9
0.96
0.96 repeat
On the table we can see various tissue samples and their absorbance values based of a
spectrophotometric reading. The ranges generally fall between 0 and 2 based off the 260/280
and 260/230 ratios. If we look at sample 883 050, we can see that the sample is a high quality
RNA sample, because the 260/230 ratio is 2.1 and the 260/280 ratio is 1.81. Both of these
values satisfy the minimum cut-off of what are considered high quality RNA extraction samples.
On the other hand, sample 908 022 is not a good sample because neither ratios fit the minimum
needed factor to even be considered for interpretation. The 260/230 ratio is 0.86 and the
260/280 ratio is 1.41. With these low numbers, the RNA sample will need be extracted and
purified again to produce better quality data in order analyze the specific RNA messenger genes.
Confirmation of RNA Quality with Gel
The other method of analyzing RNA samples is by using gel electrophoresis. The gel
electrophoresis provides a way for us to differentiate which nucleotide bands were collected
during the extraction process. The various messenger RNAs can be identified by the distance
the bands travel from the sink of concentrated RNA sample.
To prepare the RNA samples for gel electrophoresis, we must prepare the solution by dyeing
the RNA samples with micro-dye so that we can differentiate between the bands and the gel
after a current is used on the samples. While preparing the RNA samples for the gel, the
concentration must also be taken into account. Putting too much or too little RNA will make the
sample hard to decipher after running the genes under a current. The optimal range of RNA
concentration is 200 to 500 ug of total mass for RNA. This is enough to adequately identify gene
bands in the gel. After running the genes through a current, there will be separate bands that we
can distinguish to identify the specific RNA messenger that we are looking for.
S.J. Kanchanavaleerat 5/7/2015
Above is a set of samples of RNAs that have been run through an electrophoresis gel. Green
checks indicate that we have a quality read RNA, red X’s indicate we have bad data, and blue
H’s indicate we have put in too high of a concentration of RNA into the gel. Looking at the data,
we can see that some samples have definitive lines that show specific coded genes. These are
marked with green check marks because we are able to identify the gene specifically.
Meanwhile other samples in the above picture show very faint lines that are harder to
distinguish. This means we either have bad data, or must run the gel again with an adequate
concentration to better read the RNA extraction data. And lastly, there are larger black globs on
some of the gel runs. This results when we exceed 500 ug of RNA sample, which makes the
identity of the gene very hard to interpret because there is no separation between the bands to
tell them apart.
Conclusion
To obtain RNA samples for interpretation, we go through a process called RNA extraction. The
RNA extraction isolates RNA from our tissue sample through PreWash and Wash Buffer
methods in order to rinse out any extra unwanted content. We also break down the DNA within
our sample by using DNase and discard the flow-through from the collection tube. After
acquiring our data, we must evaluate our RNA sample by assessing its quality. We did this by
performing a RNA gel using a bioanalyzer to determine the genetic material within our RNA
sample.
S.J. Kanchanavaleerat 5/7/2015
References
1. "The Basics: RNA Extraction." Thermo Fisher Scientific. 2015. (7 May 2015).
https://www.lifetechnologies.com/us/en/home/references/ambion-tech-support/rnaisolation/general-articles/the-basics-rna-isolation.html
2. "RNA Isolation for Frozen Mouse liVers and Reverse Transcription."
https://www.lifetechnologies.com/us/en/home/references/ambion-tech-support/rnaisolation/general-articles/the-basics-rna-isolation.html
3. Direct-zol RNA MiniPrep. Instruction Manual. Zymo Research, 1-10.
4. “Interpreting Nanodrop (Spectrophotometric) Results. (13 May 2015).
http://www.u.arizona.edu/~gwatts/azcc/InterpretingSpec.pdf