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Use of Internal and External
Standards or Reference RNAs for Accurate Quantitation of RNA
Levels
Introduction
Northern blotting, nuclease protection
assays and RT-PCR are frequently used to analyze gene expression.
While qualitative results from these assays are sometimes sufficient,
most researchers require quantitative results.
Quantitative data can be expressed in either
relative or absolute terms. For relative quantitation, RNA levels
of the gene under investigation are compared from sample to sample
using an internal control to normalize for differences in sample
concentration and loading. Ideally, the internal control should
be a gene expressed at a constant level across the sample set.
In absolute quantitation, signal intensities representing the
expression levels of the experimental gene are compared to a
standard curve generated by samples containing a concentration
titration of an external standard.
In this technical bulletin, we describe
relative and absolute quantitation applied to Northern blotting,
nuclease protection assays and RT-PCR. We also discuss how to
choose an internal control for relative quantitation.
Relative Quantitation
In relative quantitation, RNA levels of the gene
of interest are compared from sample to sample using an internal
control to normalize for differences in sample concentration and
loading. As the internal control should be expressed at a constant
level across all samples, both choosing and authenticating this
control are critical to successful relative quantitation. In the
paragraphs below, we describe commonly used internal controls and
how these are authenticated.
Internal Standard RNA Probes
Inconsistencies in RNA isolation and in
the commonly used RNA analysis procedures listed above can introduce
errors into the analysis process. One method for minimizing these
errors is to simultaneously measure a cellular RNA that has a
constant expression level between samples. This RNA serves as
an internal standard or reference against which other RNA values
can be normalized. This process corrects for sample to sample
variation.
The ideal RNA species for an "internal
standard" should be expressed at a constant level across all
of the types of samples being analyzed. For example, the internal
standard should be expressed equally among different tissues
of an organism, at all stages of development, and for both control
and experimentally treated cell types. A constituitively expressed "housekeeping" gene
would appear to be a good model for an internal standard RNA.
Unfortunately, there is no one single RNA with a constant expression
level in all of these situations (although 18S rRNA appears to
come close to being an ideal internal control under the broadest
range of experimental conditions, see below). It is therefore
necessary to identify the appropriate control RNA for the particular
set of experimental RNA samples to be studied.
ß-actin
ß-actin mRNA was one of the first RNAs
used as an internal standard. It encodes a ubiquitous cytoskeleton
protein and is expressed at moderately abundant levels. In a
10 µg sample of total RNA, there is approximately 300 pg
of ß-actin mRNA, representing 0.1% of mRNA or 0.003% of total
RNA. This is compared to 0.3–3 pg of a rare mRNA species.
The ß-actin gene is highly conserved in eukaryotes (1) and expressed
in most cell types (2). However, the level of expression has
been shown to vary in some tissues, including cultured adipocytes
(3), mammary epithelial cell lines, breast fibroblast cell lines
(4), and cultured human colon carcinoma cells (5). Experiments
at Ambion have shown ß-actin mRNA levels to be high in normal
mouse spleen, brain, embryo, heart, hypothalamus and kidney and
relatively lower in mouse liver, lung and muscle (Figure 1).
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Figure 1. Expression
Level of Several Common Internal Standards Compared across
Mouse Tissues Using a Ribonuclease Protection Assay. High
specific activity antisense RNA probes (1.15 x 109 cpm/µg)
to GAPDH, ß-actin and cyclophilin were synthesized
from Ambion's pTRI-GAPDH, pTRI-beta-actin, and pTRI-cyclophilin
mouse DNA templates, respectively, in a MAXIscript in
vitro transcription reaction. 4 x 104 cpm
of each probe was added to 5 µg total RNA from
different mouse tissues and processed in a multiple probe
ribonuclease protection assay using the RPA II Kit. Results
show a comparison of the relative expression levels of
each probe within a given tissue and across mouse tissues.
Control lanes of yeast RNA and probe +/- RNase are shown
on the left and Ambion's Century Markers provide molecular
weight standards on the right.
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GAPDH
Glyceraldehyde-3-phosphate-dehydrogenase
(GAPDH) is a key enzyme in glycolysis. The mRNA encoding GAPDH
is another moderately abundant message in cells, with expression
levels similar to that of ß-actin. This housekeeping gene is
constitutively expressed in many tissues and has been reported
to be a useful internal control. GAPDH is expressed at high levels
in rat and human muscle and heart (6) and has been used frequently
as a control in experiments using cell lines (7, 8, 9). However,
GAPDH levels vary with developmental stage and with dexamethasone
treatment in embryonic chick heart and tendon cells (10). Furthermore,
GAPDH mRNA levels varied significantly in several different virally-transformed
or oncogene-transfected mouse fibroblast cell lines (11), during
the cell cycle of normal human skin fibroblasts (12), in rat
vascular smooth muscle cells treated with vasoconstrictor or
mitogens (13) and some breast cell lines (4). GAPDH RNA levels
have also been shown to increase significantly in cultured adipocytes
treated with insulin (14). Research at Ambion has shown GAPDH
to be expressed at relatively low levels in normal mouse liver,
spleen, lung, embryo and hypothalamus and at relatively high
levels in mouse brain, muscle, heart and kidney (Figure 1).
Cyclophilin
A third constituitively expressed gene,
cyclophilin, encodes a ubiquitous cytoplasmic protein that plays
a role in protein folding through the isomerization of peptide
bonds. It is also known to bind cyclosporin-A and may mediate
the drug's immunosuppressive effects (15). Cyclophilin mRNA is
also somewhat abundant, comprising close to 0.1% of cytoplasmic
mRNA, and is highly conserved across different species. It is
expressed at high levels in normal rat brain, spleen, thymus,
adrenal glands, ovaries and testes; however, lower level expression
is seen in rat liver, lung and anterior pituitary (16). Cyclophilin
is also expressed at relatively high levels in many monkey tissues
except skeletal muscle (16). Some tumor cell lines have shown
a higher concentration of cyclophilin mRNA than normal cells
(15). However, no alteration of cyclophilin levels have been
observed when T-lymphocytes are treated with mitogens and tumor
promoters (15). Ambion researchers have seen cyclophilin mRNA
expressed at similar high levels in normal mouse brain, thymus,
ovary, kidney and embryo and relatively lower levels in mouse
liver, heart, lung, spleen and testes (Figure 1). When compared
with ß-actin and GAPDH, cyclophilin mRNA is less abundant across
all mouse tissues tested (Figure 1). Given this fact, cyclophilin
may prove to be a more appropriate internal control in multiprobe
assays when studying the expression of rare mRNA species.
rRNAs
18S and 28S ribosomal RNAs may also be
used as internal controls. These RNAs are less likely to fluctuate
under conditions that affect the expression of mRNAs. This is
because they make up 80% of a total RNA sample, such that when
the concentration of a total RNA sample is determined from spectrophotometric
readings, the sample is essentially already being normalized
to the amount of rRNA it contains. rRNAs are also transcribed
by a distinct polymerase from mRNAs, which may result in a different
pattern of regulation of expression. Specifically, the expression
levels of 28S rRNA have been observed to remain stable while
levels of ß-actin and/or GAPDH vary. This has been seen
in individual rat livers (17), normal human skin fibroblasts
(12), malignant mouse cell lines (11) and some breast cell lines
(4). At Ambion, we have seen 18S levels to be uniform in all
mouse tissues tested. These include liver, brain, thymus, heart,
lung, spleen, testes, ovary, kidney and embryo (Figure 2). In
a 10 µg total RNA sample, 2 µg are contributed by
18S rRNA, and 5.5 µg are contributed by 28S rRNA. Thus,
if the researcher is defining an equivalent amount of RNA from
two samples to be a constant mass amount (10 µg), then
by definition, the ribosomal RNAs will be constant from 10 µg
sample to 10 µg sample. (Note that in some situations it
is more relevant to normalize RNA to a constant cell number or
tissue mass.) The very high abundance of the rRNAs requires the
researcher to generate a large amount of low specific activity
antisense probe for detecting these RNAs. (Note that the exact
amount of probe needed will depend on the size of the probe:target
region of hybridization; 0.5–1 µg of probe containing
a 100 nt rRNA sequence is needed to be in 5 molar excess of rRNA
target when performing a nuclease protection assay on a 10 µg
total RNA sample.) A few micrograms of low specific activity
probe can be synthesized using Ambion's MAXIscript™ In
Vitro Transcription Kits, but for large numbers of samples,
this is most conveniently done using the MEGAshortscript™ Large
Scale In Vitro Transcription Kit.
Of course, all of these standards described
here have not been tested in every possible system. Therefore,
it may be prudent to test more that one internal control to ascertain
which is most appropriate for a given experimental system (see "Validating
Your Internal Control", below).
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| Figure 2. Expression
Level of 18S rRNA Compared across Mouse Tissues Using a
Ribonuclease Protection Assay. A
low specific activity antisense 18S ribosomal RNA probe
was synthesized from Ambion's pT7-18S DNA template in a
MAXIscript™ in vitro transcription reaction. 3 x 104 cpm
of probe was added to 3 µg total RNA from different
mouse tissue processed in a ribonuclease protection assay
using the RPA II™ Kit. Results show the relative expression
level of 18S rRNA across mouse tissues. The doublet represents
partial renaturation of protected fragment due to high
concentration of rRNAs. Signal intensity is best obtained
by measuring both bands (see Ambion's T7-18S Antisense
DNA Specification Sheet for further explanation). Control
lanes of yeast RNA and probe +/- RNase are shown on the
right, and Ambion's Century™ Markers provide molecular
weight standards (only the 100 nt band is seen) on the
left. |
Validating Your Internal Control
Determine the validity of an internal control for your experimental system
by doing the following pilot experiment. Replicates of each sample
type (e.g., treated vs. untreated, different tissue or cell types)
will be used to analyze the expression of the potential internal
control.
Using 3 to 4 replicates will provide statistically
significant results. Add the same amount of internal control
probe or primers to each sample. Do the experiment, and evaluate
the results. Differences seen between samples should not be greater
than differences seen within replicates of each sample. Figure
3 shows a Northern experiment used to validate ß-actin
as an internal control for samples from various tissues. The
data suggest that ß-actin could be used as an internal
control for comparing samples from embryo and thymus, or heart
and liver, but that it is clearly not an appropriate internal
control for comparing samples from embryo and heart, for example.
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| Figure 3. Authenticating
an Internal Control. Total
RNA (2.5 µg) from the indicated tissues was run on
a denaturing agarose gel in quadruplicate. Panel A shows
the gel stained with Ethidium bromide. The gel was then
Northern blotted and hybridized with a radiolabeled probe
for ß-actin. The autoradiogram is shown in Panel
B. |
Relative Quantitation Using
Northern Analysis
Relative quantitation of gene expression
by Northern analysis involves multiple stripping and reprobing
cycles on the same blot. The blot should first be probed with
the least abundant message to be quantitated. The blot should
then be hybridized with the other probes in order of increasing
abundance and finally, hybridized with the internal control (most
internal controls are abundant mRNAs).
After hybridizing with the internal control,
the samples are standardized and mRNA quantitation is expressed
in relative terms. For example, in Figure 4 the liver lane has
a 0.5:1 ratio of S15: internal control signal, whereas in the
spleen lane, the ratio is 1.5:1. This indicates that S15 is expressed
at a 3X higher level in spleen.
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| Figure 4. Relative
Quantitation in a Northern. Five µg
of various mouse total RNAs were electrophoresed and transferred
to a membrane. The membrane was then hybridized with two
RNA probes - one complementary to the internal control,
GAPDH, and the other to ribosomal protein S15. The blot
was washed and exposed to film. |
Relative Quantitation Using Nuclease Protection
Assays (NPAs)
The ribonuclease protection assay (RPA)
is particularly useful for the simultaneous quantitation of multiple
mRNA species in one sample. Using Ambion's RPA III™ Kit, up to
12 different mRNAs can be detected and quantified in a single
sample. Probes used for multiprobe analysis must be of significantly
different size to allow resolution on a gel and cannot contain
regions of intermolecular complementarity. Prior to analysis,
each probe must be tested to ensure that after digestion, it
produces a single band. One of the probes must be an invariant
internal control to which the samples will be standardized.
High specific activity probes should be
used for rare targets, and low specific activity probes are needed
for moderately and highly expressed targets such as GAPDH, ß-actin
and 18S rRNA. Altering the specific activity of probes reduces
their signal intensity so that rare and abundant transcripts
can be detected with a single exposure.
Figure 5 shows the varied expression pattern
of 4 oncogene mRNAs across different tissue types. In this case,
cyclophilin is used as an internal control to normalize signal
within each of the samples.
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| Figure 5. Ribonuclease
Protection Assay Using Mouse Tissues Stored in RNAlater™. Various
mouse tissues were stored in RNAlater for 1 or 4
weeks at 4°C. RNA was isolated from each tissue and
analyzed using the RPA III™ Kit. Ten µg of total
RNA was hybridized with 5 x 104 cpm of each
of 5 combined antisense RNA probes, digested with RNase
and precipitated. Products were assessed on a 5% polyacrylamide/8
M urea gel and exposed to film for 4 hours at -80°C
with an intensifying screen. |
Relative Quantitation Using RT-PCR
Relative RT-PCR uses primers for an internal
control that are multiplexed in the same RT-PCR reaction with
gene specific primers. Internal control and gene specific primers
must be compatible; that is, they must not produce additional
bands or hybridize to each other. Additionally, for relative
RT-PCR data to be meaningful, the PCR reaction must be terminated
when the products from both the internal control and the gene
of interest are being amplified within the linear range.
Ambion recommends using 18S rRNA as an
internal control; however, because of its abundance, rare message
products could not previously be detected in the linear range
of 18S rRNA. Ambion's QuantumRNA™ 18S Internal Standards
solve this problem. By mixing primers for 18S rRNA with Ambion's
exclusive competimers — primers of the same sequence but
that cannot be extended — the 18S rRNA signal can be reduced
even to the level of rare messages.
Ambion has over 90 Gene Specific Relative
RT-PCR Kits available that contain primer pairs for specific
human, mouse, and rat genes and QuantumRNA 18S rRNA internal
control primers. Typical data generated using these kits is shown
in Figure 6.
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| Figure 6. Multiplex
Quantitative RT-PCR with Ambion's Gene Specific Relative
RT-PCR Kits. 1X (A) or
10X (B) amounts of RNA were reverse transcribed with the
RETROscript™ Kit and random decamers. Individual PCR
reactions were performed with the indicated Gene Specific
Relative RT-PCR Kit, each reaction containing gene-specific
primers, 18S rRNA primers and 18S rRNA Competimers™.
Competimer technology attenuates 18S rRNA amplification
efficiency so that it can be multiplexed effectively with
the much less abundant interleukin targets. |
Absolute Quantitation
Absolute quantitation measures
the absolute amount (e.g. 5.3 x 105 copies) of a specific
sequence in a sample. Dilutions of a synthetic sense strand RNA
(identical or very similar to the target template) are co-amplified
or detected along with the endogenous target. The sense RNA creates
a concentration curve to which the endogenously expressed message
is then compared in order to obtain an absolute measurement of
the transcript under study.
Absolute Quantitation Using
Northern Analysis
Absolute quantitation by Northern
analysis is straightforward. A sense strand RNA transcript
complementary to the probe is synthesized (see Ambion's MAXIscript™ in
Vitro Transcription Kit). The synthetic RNA is quantitated,
and dilutions are spiked into a yeast RNA background and
size fractionated in a denaturing agarose gel next to the
sample RNAs. Alternatively, a longer or shorter sense RNA,
sharing target sequence with the endogenous message, can
be added to the experimental samples. After blotting the
membrane is probed with an antisense probe, and the amount
of endogenous target is compared with the concentration curve
generated by the synthetic sense strand dilutions.
Figure 7 illustrates analysis
of ß-actin expression in various mouse tissues. For example,
comparing the expression of ß-actin in mouse liver to
the standard curve derived in this experiment shows that there
are approximately 30 pg of ß-actin RNA/µg of total
mouse liver RNA.
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| Figure 7. Quantitation
of Variable ß-actin Expression in Mouse Tissues. Mouse
total RNA from several tissues was run on a Northern gel
and transferred to membrane using Ambion's NorthernMax™ Kit.
Dilutions of full-length ß-actin sense RNA were spiked
into a yeast background and also run on the same gel. A 32P-labeled
antisense ß-actin probe was used to detect ß-actin
mRNA in the mouse tissues as well as the artificial sense
strand. Absolute expression levels were determined by direct
comparison of the signal from the mouse tissues with the
concentration curve generated with artificial sense strand ß-actin. |
Absolute Quantitation Using Nuclease Protection
Assays
As with absolute Northern analysis, the
quantitation curve for absolute NPA analysis is constructed by
spiking quantitated sense strand RNA dilutions into a yeast RNA
background. These control reactions are then processed along
with the sample RNAs and the reaction products are separated
on a denaturing polyacrylamide gel.
Figure 8 shows an example of absolute quantitation
using NPA analysis. In this experiment, approximately 10 pg of
CAT mRNA are expressed per 100,000 pSVCAT transfected COS cells.
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| Figure 8. Detection
of Cloramphenicol Acetyltransferase (CAT) Expression in
COS Cells Transiently Transfected with pSVCAT. Increasing
dilutions of pSVCAT-transfected COS cells were assayed
for CAT by ribonuclease protection. Reaction products were
resolved on a denaturing 8% polyacrylamide gel, dried,
and autoradiographed. Lane 1, CenturyĆ Markers; lane 2,
undigested CAT probe; lane 3, CAT probe digested after
hybridization with total RNA from 4 x 105 COS
cells; lanes 4-9, CAT probe digested after hybridization
with total RNA from 4 x 105, 2 x 105,
105, 5 x 104, 2.5 x 104,
and 1.25 x 104 transfected COS cells; lanes
10-14, standard concentration curve generated by digestion
of the CAT probe after hybridization to 10, 20, 50, 100,
and 200 pg of truncated sense transcript diluted in 5 µg
of yeast RNA. |
Absolute or Competitive RT-PCR
Competitive RT-PCR precisely quantitates
a message by comparing signal intensity of the RT-PCR product
to a concentration curve created using a synthetic transcript
or "competitor". Competitors are designed to:
- be amplified using the same primers
as the desired target sequence,
- have the same amplification efficiency
as the target sequence,
- be of a significantly different size
from the target to allow differentiation of the two products
on an agarose gel, and
- control for variations in the RT reaction.
Competitor
RNA transcript is synthesized, quantitated and diluted into the
sample RNA. Pilot experiments are run to determine the range
of competitor concentration where the experimental signal is
similar in concentration to the endogenous target. A more precise
concentration can then be determined by repeating the experiment
with a smaller dilution range, generating data that approximates
abundance of the endogenous target in the sample.
From the data illustrated in Figure 9,
it can be determined that between 1.5 x 106 – 3.05
x 106 copies of hIL-10 RNA are present in the sample.
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| Figure 9. Competitive
RT-PCR Experiment. The
indicated amounts of hIL-10 Armored RNA® competitor
were added to 2 µg aliquots of experimental RNA,
and the mixture underwent RT-PCR. |
Products Available
Ambion offers a variety of internal standards
as linearized plasmid templates to generate antisense RNA or DNA
probes by in vitro transcription or primer extension. These include
human, rat and mouse templates for ß-actin,
GAPDH and cyclophilin and regions of 18S and 28S rRNA that cross-react
with most vertebrates with few, if any, mismatches (Ambion's 18S
rRNA template can be used with virtually any eukaryote). All transcription
templates (except pT7-18S-rRNA) are inserted into our unique pTRIPLEscript™ vectors.
pTRIPLEscript vectors feature three tandem phage promoters, which
offer the researcher the convenience of synthesizing an antisense
RNA transcript using SP6, T7 or T3 polymerase. They are ideal for
the synthesis of antisense internal control standards and can be
used in Northern blots or as a second probe in ribonuclease and
S1 protection assays for simultaneous detection of both an internal
reference RNA and the mRNA being studied. Ambion also provides
DECAtemplates™ for making random-primed DNA probes for commonly
used internal controls (18S rRNA, ß-actin,
GAPDH, and cyclophilin).
Along with our line of internal control templates,
we offer an extensive line of transcription templates for human
and mouse oncogene studies as well as high-quality RNA for RNA
expression analysis. Ambion's MAXIscript™ Kits are ideal for
the transcription of high specific activity antisense probes (the
MEGAshortscript™ Kit is another option for transcribing large
amounts of antisense rRNA probes). RPA II™ and RPA III™,
and HybSpeed™ RPA Kits are
also available for quick and accurate analysis of RNA samples.
For accurate RT-PCR analysis, we provide the QuantumRNA™ family
of kits (with primer and Competimer™ sets for 18S rRNA and ß-actin)
for relative quantitation.
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