Quality assessment of total RNA








RNA quality control using the NanoDrop ND-1000

There are three quality controls that are performed on isolated RNA. One is to
determine the quantity of RNA that has been isolated, the second is the purity of RNA
that has been isolated and the third is the integrity of the RNA that has been isolated.



Measuring the Quantity of RNA using the Nanodrop.

Nucleic acids are traditionally quantified using UV absorption using a
spectrophotometer.
In its simplest form the absorbance is measured at 260 and 280 nm. The
concentration of nucleic acid can be determined using the Beer-Lambert law, which
predicts a linear change in absorbance with concentration.

An A260 reading of 1.0 is equivalent to about 40 µg/ml of RNA and the OD at 260 nm
is used to determine the RNA concentration in a solution.

RNA has its absorption maximum at 260 nm and the ratio of the absorbance at 260
and 280 nm is used to assess the RNA purity of an RNA preparation. Pure RNA has
an A260/A280 of 2.1. You will see in many protocols that a value of 1.8-2.0 indicates
that the RNA is pure. This depends, however on how you performed the
measurement and the source of putative contaminations.

Ideally, scanning spectrophotometry should be used as this makes it possible to
also identify possible sources of contamination. One of the problems using
conventional spectrophotometers is that the cuvettes are large making it difficult to
measure low concentrations of RNA without losing an unacceptable fraction of the
sometimes precious and valuable RNA sample.
The NanoDrop® ND-1000 UV-Vis Spectrophotometer enables highly accurate
analyses of extremely small samples with remarkable reproducibility. The sample
retention system eliminates the need for cuvettes and capillaries, which decreases
the amount of sample required for the measurement.

Surface tension is used to hold a column of liquid sample in place while a
measurement is made. This is done by pipeting the sample (only 1 or 2 micro liters!!)
directly onto one measurement pedestal. A measurement column is then drawn
between the ends of two optical fibers to establish the measurement path as shown
in the figure. The measurement is made, typically in less than 10 seconds, and the
spectrum and its analysis is shown on the screen of the attached PC and archived on
the PC. Once the measurement is complete, the sample is simply wiped from the
measurement pedestals. The archived data
can be manipulated by a spreadsheet program such as MS Excel.

Using the Nanodrop will provide you with a scan of the absorbance from about 200
nm up to 350 nm, which is the relevant region for determining RNA concentration and
purity.












































As mentioned above, RNA has its absorbance maximum at 260 nm and this
absorbance is not dependent on the pH of the solution. However, the absorbance of
some of the contaminants (like proteins) in the RNA solution have an absorbance
that is pH-dependent. This means that although the A260 reading of the RNA solution
with remain the same at different pHs, the A280 reading will differ at a pH dependent
manner.

It has been shown that significant variability in the A260/A280  ratio can occur when
different sources of water were used to perform the spectrophotometric
determinations. Adjusting the pH of water used for spectrophotometric analysis from
approximately 5.4 to a slightly alkaline pH of 7.5-8.5 significantly increased RNA
A260/A280  ratios from approximately 1.5 to 2.0.

Wilfinger WW, Mackey K, Chomczynski P., Effect of pH and ionic strength on the
spectrophotometric assessment of nucleic acid purity. Biotechniques. 1997, Mar;22(3):
474-6, 478-81.

Consequently, if you measure your RNA in pure water, and you have an OD
A260/A280  ratio of 1.8, your RNA quality might be pure but you will not be able to
exclude that it is contaminated with DNA, protein or something else. Measuring RNA
in a buffered solution like TE (pH 8.0) will result in an OD A260/A280  ratio that is
more reliable. If you measure RNA in TE (pH 8.0), you should get an OD A260
reading very close to 2.0. If not, your sample is contaminated.

The same sample measured in pure water that gives you an OD of maybe 1.5-1.8
could give you an OD ratio of 2.0 in TE pH 8.0.

As can be seen from the figure, measuring RNA absorbance in water and diluting it
twofold for several times will result in a decrease of the A260/A280 ratio. Performing
the measurement in TE (pH 8.0) results in exactly the same OD A260/A280  for all the
dilutions.


Besides the pH, the A260/A280  ratio is also dependent of the ionic strength of the
spectrophotometric solution. Therefore, it is important to use exactly the same buffer
as a diluent and as the blank, sometimes pure water is used incorrectly as a blank.
This is in particular a problem if you do not know the composition of for example the
elution solution that comes with a commercial RNA isolation kit.

Most protocols only mention the OD A260/A280  ratio but you will often see in the
region of 230 nm a strong absorbing contaminant. Let there be no mistake, this is not
RNA as pure RNA has a single peak with a maximum at 260 nm. There are three
sources of contaminations that produce peaks in the 220-230 nm region, these are
proteins, chaotropic salts like guanidinium isothiocyanate and phenol. All these three
are either present in your tissue sample or are present in most tissue lysis solutions
derived from the original Chomczynski and Sacchi protocol.

Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium
thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156-9.  

To our opinion, it is important that not only the OD A260/A280  ratio should be very
close to 2.0, but that in addition, also the OD A260/A230  ratio should be very close to
2.0. Specially, when isolating low amounts of RNA the OD A260/A230  ratio drops
significantly to sometimes under the 1.0. This clearly indicates, contamination with
chaotropic salts or rests of phenol or protein in the RNA solution.
RNA QUALITY CONTROL
Effect of several frequently used reagents on the absorbance of RNA
Effect of ethanol contamination
Effect  of isopropanol contamination
Effect of TRIS contamination
Effect of protein contamination
Effect of guanidine isothiocyanate contamination
Serial dilution of RNA 200-20 ng/µl in pure water (left panel) or in TE pH8 (right panel). This
figure shows the effect of measuring RNA absorbance in buffered TE, pH 8.0 or non-buffered
solutions (pure water). The left panel shows the results of measuring in pure water. It is clear
that the same RNA concentrations give comparable values in water or TE pH 8.0 as the
absorbance of RNA at 260nm itself is independent of pH. However, measuring in TE pH 8.0
results in significantly higher OD 260/280 ratios as the absorbance of contaminants is pH
dependent.
The OD A260/A280 for the 25 ng/µl RNA in pure water is 1.81, while it is 2.04 in TE
.
Minor contamination problems: TRIS, ethanol and isopropanol
Figure 1.
This figure shows that TRIS contamination does not influence the absorbance spectrum
of RNA significantly. As TRIS is a buffer by itself, in this case there is also no
difference between measuring in water (left panel) or TE pH8.0 (right panel).
Figure 2.
This figure shows that isopropanol does not have a significant effect on the absorbance
spectrum of RNA when measured in TE pH 8.0. However, when the measurement in made
in pure water, the OD A260/A280 ratio of 1.82 at 0.5 % isopropanol contamination is
reason for serious doubt on the quality of the RNA. In TE the OD A260/A280 ratio is 2.10
indicating high purity.
Figure 3.
This figure shows that ethanol does not have a significant effect on the absorbance
spectrum of RNA when measured in TE pH 8.0. However, when the measurement in made in
pure water, the OD 260/280 ratio of 1.76 at 0.5 % ethanol contamination is reason for
serious doubt on the quality of the RNA. In TE the OD A260/A280 ratio is 2.06 indicating
high purity.
Major contamination problems: protein, guanidine isothiocyanate and phenol
Figure 4.
In this figure the effect of protein contamination can be seen.  A contamination level of 0.01 %
BSA (upper panels) shows an almost normal absorbance spectrum although the OD A260/A280
ratio is below 1.9, which is a warning that something is contaminating the sample. At 0.5 % BSA
(lower panels), the absorbance spectrun looks awful and this is a clear indication that there is a
significant level of contamination in these samples.
Figure 5.
This figure shows the dramatic effect that
guanidine isothiocyanate has on the absorption
spectrum of RNA. Note that guanidine
isothiocyanate contrary to phenol hardly
influences the OD A260/A280 ratio, which in
these examples is still well above 2.0.
Therefore, Guanidine isothiocyanate has a
small effect on the quantification of RNA.
Guanidine isothiocyante on the other hand, has
a very strong effect on the OD A260/A230 ratio,
which at the 0.5 % contamination level drops
below 0.5. An OD A260/A230 below 1 should
be avoided as the level of guanidine might have
a deleterious effect on downstream enzymatic
reactions.
Figure 6.
This figure shows the dramatic effect that phenol can have on the absorbance spectrum of RNA.
Phenol has a very strong effect on the quantification of RNA. Note that at a 0.5 % contamination
level, the measured concentration is over three times as high as the actual value of 50 ng/µl.
This strong disturbing effect of phenol on the quantification of RNA is due to the contaminating
absorption peak at 270 nm. At very low RNA concentrations below 10 ng/microliters this
contaminating peak is often even confused for RNA. It cannot be stated more clear: THE RNA
ABSORBANCE PEAK IS NEVER AT 270 nm. If the peak is at 270 nm it arose from a
contamination. At low RNA concentration after the RNA has been isolated using a method based
on phenol extraction, the erroneous overestimation of RNA levels is a serious problem in RNA
work.
Effect of phenol contamination
The Good
Both the OD A260/A280 as the OD
A260/A230 ratio are 2.0 or more.

Perfect, you can do with this RNA
whatever you like, everything should
work.
The Ugly
Don’t even think of using this RNA!
Just perform an extra purification step
.
The Bad
The OD A260/A280 ratio is over 2.0 but the
OD A260/A230 ratio is below 1.0. Be
careful! This indicates that the sample
contains impurities. Some downstream
procedures may work perfectly while others
may give problems.
The Good, The Bad and the Ugly

Here below you will see some RNA absorbance spectra of RNA that is pure, and
also of RNA for which you should be able to conclude that the sample is not pure. In
order to help you identify possible sources of contaminations, we have spiked pure
RNA with the most commonly used reagents used for the RNA extraction and
purification procedures. Hopefully, this may be helpful for you to identify your source
of contamination if it interferes with your specific downstream processes.

When you make an absorption spectrum of your RNA sample with the Nanodrop,
you will be able to save your data and import them into an Excel spreadsheet. In this
way you will be able to make composite figures and compare the results obtained
from several purification procedures or the effects of contaminants on the
absorbance of RNA.
Figure 7.
This figure shows the effect of measuring absorbance of RNA in non-buffered water or in TE
pH 8.0.  Note the increase in absorbance at 280nm in the non-buffered solution which results
in a lower OD 260/280 ratio.
Figure 8.
This figure shows the effect of protein contamination on the RNA absorbtion. Note that protein
decreases the absorption both at 260 and at 280 nm so that the net results is a decrease in the
OD 260/280 ratio well below 1.9.
Figure 9.
This figure shows that EDTA contaminations will not significantly disturb OD measurements at
260 and 280 nm. Also alcohols like ethanol and propanol do not have significant effects at low
contamination levels of a few percents (V/V).
Figure 10.
This figure shows the dramatic effect of guanidine isothiocyanate on the absorbance of RNA.
Note the large absorbance below 230 nm.
Figure 11.
This figure shows that RLT, which is a buffer used in the Qiagen RNeasy  RNA isolation kits
also contains a guanidine isothiocyanate-like component that may interfere significantly with
RNA OD measurements. Note also for RLT the large absorbance below 230 nm.
Figure 12.
This figure shows the effect of Trizol on the absorbance of RNA. Note that Trizol also contains
guanidine isothiocyanate that strongly absorbs below 230 nm. In addition, Trizol contains phenol
which is responsible for the strong absorption at 270 nm.
Figure 13.
A composite figure showing relative effects on frequent RNA contaminations. GIT: Guanidine
isothiocyanate, RLT: buffer used in Qiagen RNeasy kits, Trizol contains both guanidine
isothiocyanate and phenol. Phenol is responsible for the large absorbance at 270 nm.
The NanoDrop ND-1000 enables the analysis of 1 ul samples, and also eliminates the
need for cuvettes and capillaries.
With the sample apparatus open, a
droplet of sample is pipetted onto the
measurement pedestal. When the sample
apparatus is closed, the sample arm
slightly compresses the droplet and a
sample column is drawn. Surface tension
alone holds the sample in place.
When the measurement is
complete, the sample
apparatus is opened and the
sample is simply wiped from
both the sample arm and
sample pedestal using an
ordinary dry laboratory wipe.
Dose response effects of common contaminators on RNA absorbance
Copyright © 2007 Website created by Mariam Ayubi
Please report problems to
admin@biomedicalgenomics.org
The invaluable help of Bieke Vanherle, Erika Timmer and Paul Wackers
is acknowledges in performing the experiments
described in this section.
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