Quality assessment of total RNA

(from http://biomedicalgenomics.org/index.html)

RNA quality control: NanoDrop ND-1000

RNA quality control: Agilent's 2100 Bioanalyzer

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 g/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 loosing 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 1-2 microliters) 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) will result in an OD A260/A280 ratio that is more reliable. If you measure
RNA in TE (pH 8), 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.
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) 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.

Serial dilution of RNA 200-20 ng/l in pure water (left panel) or in TE pH 8 (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 260 nm 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/l RNA in pure water is 1.81, while it is 2.04
in TE

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.

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 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 Ugly
Dont even think of using this RNA! Just perform
an extra purification step.

Effect of several frequently used reagents on the absorbance of RNA

- Minor contamination problems: TRIS, ethanol and isopropanol

Effect of TRIS contamination: 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 pH 8.0
(right panel)

Effect of ethanol contamination: his 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.

Effect of isopropanol contamination: 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.

- Major contamination problems: protein, guanidine isothiocyanate and phenol

Effect of protein contamination: 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
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that something is contaminating the sample. At 0.5 % BSA (lower panels), the absorbance
spectrum looks awful and this is a clear indication that there is a significant level of
contamination in these samples.

Effect of guanidine isothiocyanate contamination: This figure shows the dramatic effect
that guanidine isothiocyanate has on the absorption spectrum of RNA. Note that guanidin
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 isothiocyanate 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.

Effect of phenol contamination: 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/l. 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.

- Dose response effects of common contaminators on RNA absorbance

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.

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.

This figure shows the effect of protein contamination on the RNA absorption. 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.

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).

This figure shows the dramatic effect of guanidine isothiocyanate on the absorbance of
RNA. Note the large absorbance below 230 nm.

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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.

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.

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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.

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RNA quality control: Agilent's 2100 Bioanalyzer

Quality assessment of total RNA sample
Total RNA run on a denaturing agarose gel or on Agilent's 2100 Bioanalyzer shows two
distinct ribosomal peaks corresponding to either 18S and 28S for eukaryotic RNA or 16S and
23S for prokaryotic RNA and a relatively flat baseline between the 5S and 18S ribosomal
peaks. The major human rRNA species (18S and 28S) are synthesized by cleavage from a
common 13 kb transcription unit which is part of a 40 kb tandemly repeated genomic unit.
The human genome contains many hundreds of these repeated rRNA genes (335 rRNA
genes, according to NCBI Assembly 36), which when transcribed result in extremely high
levels of rRNA transcripts. There can be as many as 10 million copies of rRNA per cell. In
addition to the many hundreds of rRNA genes, the extremely high stability of the rRNA
transcripts (4-5 days) strongly contributes to their high levels within the cell.

Biosynthesis of rRNA transcripts. Small arrows indicated by letters A-D signify positions of endonuclease
cleavage of RNA precursors. Cleavage of the 41S precursor at B generates two products: 20S + 32S. Following
cleavage of the 32S precursor at D, and excision of the small 5.8S rRNA, hydrogen bonding takes place
between the 5.8S rRNA and a complementary central segment of the 28S rRNA. The approximately 6 kb of RNA
sequence originating from the external and internal transcribed spacer units (ETS, ITS1 and ITS2) are degraded
in the nucleus. S is the sedimentation coefficient, a measure of size. The figure was adapted from: Human
Molecular Genetics 2 from Tom Strachan and Andrew P. Read.

The fact that the 18S and 28S rRNA species are derived from the same precursor molecule
means that there are exactly the same number of copies of both molecules in the cell.
Traditionally, the intensity of these rRNA bands on denaturing agarose gels have been used
to calculate a ratio that served as an indication of RNA integrity. A 28S/18S ratio of two is
considered to be good quality RNA.

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Traditional separation of total RNA on denaturing agarose gel electrophoresis followed by

ethidium bromide staining. The 28S/18S RNA bands in this figure have an intensity ratio
around 2 that would be considered good to very good quality RNA.

If one would consider the amount of bases of these major rRNA species (for human, the 28S
rRNA species has 5034 bases and the 18S rRNA species has 1870 bases) the exact ratio
should be 2.7 and for mouse rRNA (see table) this should be 2.5. The reason that the 28S
rRNA band or peak is seen as more intense despite the fact that there are exactly as many
molecules as for the 18S rRNA is because the detection of rRNA depends on the binding
(intercalation) of dye to the RNA and this depends on the number of bases present in each
molecule. So twice as many bases will bind twice as many dye molecules, which will result
in a twice as intense signal intensity of the band on gel or the electropherogram peak of
the Bioanalyzer.
Table: Size of human and mouse rRNA molecules and the theoretical ratios of their 28S and 18S rRNAs.

Species
Human

rRNA
28S
18S

Size (bases)
5034
1870

Ratio 28S/18S
2,69

Mouse

28S
18S

4729
1869

2,53

The major problem with traditional denaturing gel electrophoresis is the large amount of
RNA required to detect a clearly visible band. Depending on the system used, this could be
a few hundred nanograms up to even a microgram of total RNA that would be required to
visualize the rRNA bands. It is clear that if small needle biopsies or laser capture material
is to be used, these amounts of RNA could not be used up just for checking the quality of
the RNA. In this sense, the Bioanalyzer is a great asset to the modern diagnostic lab for
those samples of which only limited amounts of sample for extracted RNA are available to
begin with.

Agilent's 2100 Bioanalyzer, Lab on a Chip for the quality control of RNA
Agilent's 2100 Bioanalyzer uses a lab on a chip approach to perform capillary
electrophoresis and uses a fluorescent dye that binds to RNA to determine both RNA
concentration and integrity. The electrophoretic analysis on the chip is based on
traditional gel electrophoresis principles that have been transferred to a chip format. The
chip format dramatically reduces sample consumption but also separation time.

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The Bioanalyzer uses two chips for the analysis of RNA. The Nano LabChip kit for the analysis of low amounts of
RNA (25 ng/l 500 ng/l) and the Pico LabChip for even lower levels of RNA (50 pg/l to 5000 pg/l). Note
that 50 picograms of RNA is the equivalent of about 5 cells if you assume 10 picograms of total RNA per cell!.
For comparison, the amount of DNA per cell is about 7 picograms.

The Bioanalyzer has a good linear dynamic range and the RNA 6000 Nano assay can be used
for RNA concentrations ranging from 25 ng/l 500 ng/l. Although the lower quantitative
limit of the RNA 6000 Nano assay is specified as 25 ng/l it is recommended to use at least
50 ng/l for a meaningful RNA Integrity Number (RIN). When using lower concentrations,
higher sample to sample variances of the RIN may be observed. However, as little as 200
pg/l of total RNA can be analyzed reliably with the Pico assay, saving most of your
valuable preps. The Pico assay can be used to assay RNA integrity in the 50 pg/l to 5
ng/l range. The figure below shows, however, that the Pico assay can detect even lower
amounts of RNA, even below 50 pg/l. These low levels of sensitivities, however, will only
be reached using extremely pure RNA!

How does Agilent's 2100 Bioanalyzer work?

The chip accommodates sample wells, gel wells and a well for an external standard
(ladder). Micro-channels are fabricated in glass to create interconnected networks among
these wells. During chip preparation, the micro-channels are filled with a sieving polymer
and fluorescence dye. Then, twelve samples (NanoChip kit), or eleven samples(PicoChip
kit) and ladder with marker are loaded in each well.
Once the wells and channels are filled, the chip becomes an integrated electrical circuit.
The 16-pin electrodes of the cartridge are arranged so that they fit into the wells of the
chip. Each electrode is connected to an independent power supply that provides maximum
control and flexibility.

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Charged biomolecules like DNA or RNA are electrophoretically driven by a voltage gradient,
similar to slab gel electrophoresis. Because of a constant mass-to-charge ratio and the
presence of a sieving polymer matrix, the molecules are separated by size. Smaller
fragments are migrating faster than larger ones. Dye molecules intercalate into RNA
strands and these complexes are detected by laser-induced fluorescence. Data is
translated into gel-like images (bands) and electropherograms (peaks).
An RNA 6000 ladder standard is run on every chip used as a reference for data analysis.
(either the RNA 6000 Nano- or Pico Ladder). The RNA 6000 ladder contains six RNA
fragments ranging in size from 0.2 to 6 kb (0.2 kb, 0.5 kb, 1.0 kb, 2.0 kb, 4.0 kb, and 6.0
kb) at a total concentration of 150 ng/l (the Pico ladder at a total concentration of 1
ng/l).
During the chip run, the dye intercalates directly with the RNA and all bands pass the
detector at different speeds. An extra lower marker fragment is run with each of the
samples to compensate for drift effects that may occur during the course of a chip run.
The software automatically compares the unknown samples to the ladder fragments to
determine the concentration of the unknown samples and to identify the ribosomal RNA
peaks.

The RNA 6000 Nano ladder (upper part) contains six RNA
fragments ranging in size from 0.2 to 6 kb (0.2 kb, 0.5 kb,
1.0 kb, 2.0 kb, 4.0 kb, and 6.0 kb) at a total concentration
of 150 ng/l. The RNA 6000 Pico ladder (lower part)
contains the same RNA fragments but at a total
concentration of 1 ng/l

How does intact total RNA looks like?

The Bioanalyzer electropherogram of total RNA shows two distinct ribosomal peaks
corresponding to either 18S and 28S for eukaryotic RNA or 16S and 23S for prokaryotic RNA
and a relatively flat baseline between the 5S and 18S ribosomal peaks.

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Good quality total RNA.

- Marker peak
- Depending on the RNA extraction method, the small 5S, 5.8S and tRNAs may be present in the
electropherogram between the marker peak and the 200 nucleotide region
- Two ribosomal peaks, the 18S and 28S rRNA peaks
- The baseline between the internal marker and the 18S rRNA peak is relatively flat and free of
small rounded peaks corresponding to smaller RNA molecules that are degradation products of
the rRNA transcripts

In general, when analyzing good quality RNA, the height of the 28S rRNA peak should be at
least twice that of the 18S rRNA peak. For perfect quality RNA we indeed find ratios over 2
with the Bioanalyzer. However, almost exclusively RNA isolated from cell culture samples
results in 28S/18S ratios larger than 2.
When isolating RNA from solid tissues, it is very difficult to get 28S/18S ratios over 2. The
main reason for this is that besides some enzymatic degradation that occurs during any
kind of RNA isolation procedure, the mechanical homogenization of the tissue on itself will
result in some RNA molecules being sheared. As the 28S rRNA molecule is more than twice
as large as the 18S rRNA molecule, it has a bigger chance to get sheared and simply
because of its larger size it is more vulnerable to mechanical disruption. This results in a
steep decline in the 28S/18S ratio when mechanical homogenization has to be used in
order to homogenize tissue samples. This is particularly a problem when tough tissues are
being used like heart, skeletal muscle or blood vessels. In addition, freezing samples will
also result in some degradation of the large RNA molecules due to ice crystal formation as
a consequence of the freezing procedure.
In the lower part of the electropherogram between 25 and 200 nucleotides, 5S RNA and
transfer RNAs can be seen because they cannot be separated on the Bioanalyzer series II
Nano or Pico assays. However, the Small RNA Assay offers a detailed view on the 6150
nucleotide range.
Typically, Trizol (Invitrogen) isolations do not remove 5S and tRNA species, while many
column-based RNA extraction kits (like Qiagen RNeasy RNA isolation kits) do remove these
small RNA species. In Eukaryotic cells the 5S/tRNA may represent up to 5 to 15 % of the
total RNA levels within a cell or tissue and this amount is tissue-specific. If micro RNAs are
to be analyzed, special care has to be taken to insure that an appropriate RNA isolation
procedure has been used that does not remove these small RNAs.

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Effect of RNA extraction procedure on low molecular weight RNAs

The RNA sample in the upper figure was extracted using only Trizol from Invitrogen, as
recommended by the manufacturer. The sample in the lower figure has been extracted by Trizol
followed by a column purification step with RNeasy form Qiagen. Note the presence of small
molecular weight RNAs in the region between the marker (25 nucleotides and the 200 nucleotide
region. These small RNAs are almost absent after the second column purification step, which is
good for your purity but it may not be what you want if you would like to analyze micro RNAs in
your sample.

The RNA Integrity Number (RIN)

Although the 28S/18S rRNA ratio is a reasonable way to estimate RNA integrity, it is not
ideal and in particular in the context of the Bioanalyzer, which does not run under
denaturing conditions sometimes the ratios do not reflect the exact level of RNA
degradation. Besides the ratio of the 18S to 28S ribosomal RNAs, Agilent has introduced a
software algorithm that takes the entire electrophoretic trace into account to help
scientists estimate the integrity of total RNA samples.
The RNA Integrity Number (RIN) software algorithm allows the classification of total RNA,
based on a numbering system from 1 to 10, with 1 being the most degraded and 10 being
the most intact. In this way, interpretation of an electropherogram is facilitated,
comparison of samples is enabled which ensures better reproducibility.
Although the RIN tremendously facilitates the assessment of the integrity of RNA
preparations and makes it much easier to compare samples or RNA isolation procedures, it
is not possible yet to determine ahead of time whether an experiment will work or not. For
example, a RIN of 5 might not work for a microarray experiment, but might work for an
RT-PCR experiment. In general, RINs higher that 7-8 seem to be working well in most of
our experiments. RINs below 7 require extra validation studies before we are able to
conclude how bad is still good enough".

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It is important to realize that we assume that the integrity of the rRNA species provides a
good indication for the integrity of the mRNA species, which is most of the time the RNA
species that we are interested in. Although this is a reasonable assumption, it is by no
means a fact. As rRNA transcripts should be considered stable (half-life of 4-5 days), a
significant fraction of the mRNA could be completely degraded while there still could be
enough high molecular weight rRNA left.
The suitability of the RNA sample is strongly dependent on the source of the RNA and the
application intended for it be it a qualitative or quantitative application. If for example
one would like to determine the presence of a particular exon within an mRNA isoform, it
would not matter if the total RNA would have a RIN of 1. As long as the splice variant can
be detected either by size or by sequencing the PCR fragment obtained form the sample. If
one would like to perform RT-PCR to show the presence of a particular transcript within a
cell type or tissue, again the RIN could be between 1-5 and still one would be able to
conclude that the transcript is expressed in that particular tissue. One should be careful by
concluding that the transcript was absent if the RT-PCR gave negative results and was
performed on RNA with a RIN below 5, specially if the transcript has a short half-life. For
every type of quantitative analysis, performing experiments with RNA with a RIN below 6
should be done with caution. Specially, if one would be using freshly harvested cultured
cells or freshly harvested tissue from a laboratory animal, the RIN should be definitely
higher that 7.5-8 otherwise there was a problem with the way the RNA extraction
procedure was performed. In some situations one has no choice, as when a sample has
been stored for many years in the freezer and/or if the tissue has been fixed with
formaldehyde. Isolation of RNA from these type of tissues can not result in perfect quality
RNA and every one should decide for themselves if they will use the sample and for what
purpose. Needless to say that for highly demanding (and expensive) assays like microarray
analyses, one would like to have RIN values higher that 7 for frozen tissues, but definitely
higher that 8 for tissue cultured cells.

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RNA quality assessment using the Bioanalyzer, The Good, the Bad and the Ugly.

There is just one way to look good and

a thousand ways to look bad

A collection of total RNA samples with decreasing RINs.

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More examples for RNA Integrity Numbers: The RNA Integrity Database (RINdb)
If you still would like to compare the quality of RNAs from several types of tissues or if you
would like to determine the quality of RNA extracted by several methods you may want to
search the RNA Integrity Database (RINdb).
The RNA Integrity database (RINdb) is a freely accessible collection of records containing
electropherograms, and other sample metadata. These include RNA source, extraction
method, RNA Integrity Number and downstream experiment quality data.

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Scientists can query the database to prescreen methods for a new experiment, compare
RIN thresholds and validate their own results. Users can also contribute records of their
own in order to help to enlarge the database.
RIBdb: http://www.chem.agilent.com/RIN/

Fuente:
Genome Center Maastricht
http://biomedicalgenomics.org/RNA_quality_control.html

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