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Analysis of Differential Gene Expression
Microarray analysis and differential display
have become popular techniques for identifying differentially expressed
genes. Once identified, the varying expression levels of specific
mRNAs must be confirmed. A number of widely used procedures exist
for evaluating the expression pattern of a particular mRNA across
RNA samples.
Here, we provide a review of the three most popular
methods for expression analysis: Northern blot analysis, Ribonuclease
Protection Assays (RPAs) and Reverse Transcription Polymerase Chain
Reaction (RT-PCR). In theory, each of these techniques can be used
to precisely determine the level of a specific RNA within a population.
However, in practice, each technique has inherent technical advantages
and limitations, which may make it more or less amenable to your
application.
Northern Blot Analysis
Despite the advent of more sensitive techniques,
Northern blot analysis remains the standard for detection and quantitation
of mRNA levels. In this technique, sample RNA is separated by denaturing
agarose gel electrophoresis, transferred to a solid support and
immobilized. A radiolabeled or nonisotopically labeled RNA or DNA
probe is then used to detect the message of interest. Typically,
ethidium bromide-stained or radiolabeled RNA markers are run on
the same gel as sample RNA to provide an accurate sizing ladder
in gels or on autoradiographs.
Pros
Straightforward Procedure: Technically,
Northern analysis presents several important advantages. First,
the method is "low-tech" in that it uses electrophoresis equipment
found in most molecular biology laboratories and that it requires
minimal finesse in the physical processing of samples. The RNA
undergoes very little manipulation; no enzymatic reactions or amplification
are carried out prior to analysis. Northern analysis also provides
opportunities to evaluate progress at various points, e.g. to estimate
how intact the RNA sample is and how efficiently it has transferred
to the membrane.
Information about Transcript
Size: Northern blot analysis is the easiest method for
determining both transcript size and the presence of alternatively
spliced or multiple transcripts generated from a single locus.
Quantitation: Northern
blot analysis allows a direct relative comparison of message abundance
between samples on a single blot. Absolute quantitation of a message
is also straightforward. A series of different concentrations of
an artificial sense-strand RNA target (exogenous standard) is spiked
into RNA samples to construct a standard curve against which experimental
sample signal can be compared. Details on how to set up an absolute
quantitation experiment are provided in Ambion's Technical Bulletin
#165.
Choice of Probes: Northern
blotting is also exceptionally versatile in the type of probe that
can be used for hybridization. High specific activity random-primed
or PCR-generated DNA probes, in vitro transcribed RNA probes, and
oligonucleotide probes can all be used successfully. Additionally,
probes with only partial homology (e.g., a cDNA from a different
species or fragments of genomic DNA, which might contain one or
more exons) may be used.
Cons
Intolerant of Degradation: Despite
these advantages, there are limitations associated with Northern
analysis. First, it is the most sensitive of the three techniques
to RNA degradation. If RNA samples are even slightly degraded,
the quality of the data and the ability to quantitate expression
are severely compromised. For example, a single cleavage event
in 20% of the transcripts of a 4 kb mRNA will decrease the returned
signal by 20%. Thus, RNase-free technique is essential.
Sensitivity: Northern
analysis is, in general, the least sensitive of the three techniques
described here, although improvements in sensitivity can be achieved
by using high specific activity antisense RNA probes or high performance
hybridization buffers (e.g., Ambion's ULTRAhyb® Ultrasensitive
Hybridization Buffer). Sensitivity can also be improved by using
oligo(dT)-selected RNA instead of total RNA. This partially circumvents
the physical constraints of gel electrophoresis and membrane transfer,
which limit the amount of RNA that can be loaded in a gel well.
Difficulty with Multiprobe
Analysis: To detect more than one message, it is usually
necessary to strip the initial probe before hybridizing to a
second probe. This process can be time consuming and problematic,
although use of stripable probes can simplify
and improve this procedure.
Ribonuclease Protection Assays
The Ribonuclease Protection Assay (RPA) is an
extremely sensitive method for the detection and quantitation of
specific RNAs in a complex mixture of total cellular RNA. An optimized
reaction may be 10 to 100+ fold more sensitive than Northern analysis
and it is much more tolerant of partially degraded RNA. The basis
of the RPA is solution hybridization of an antisense probe (radiolabeled
or nonisotopically labeled) to an RNA sample. After hybridization,
single-stranded, unhybridized probe and RNA are degraded by ribonucleases.
The remaining hybridized probe:target fragments are separated on
an acrylamide gel and visualized by autoradiography. If nonisotopic
probes are used, samples are visualized by transferring the gel
to a membrane and performing a secondary detection step.
Pros
Hybridization Efficiency: Solution
hybridization is far more efficient than filter-based hybridization
and does not have the limitation of maximum membrane capacity,
so an RPA reaction can accommodate up to 100 µg of total or poly(A)
RNA. This translates to increased sensitivity.
Sample Quality Requirements: RPAs
are less sensitive to partial RNA degradation than Northern analysis.
A single cleavage in 20% of a 4 kb message may cause as little
as a 1% loss of signal, since cleavage is only detected in the
region of complementarity with the probe.
Multiprobe Analysis: RPAs
are the method of choice for simultaneous quantitation of several
RNA targets. During solution hybridization and subsequent analysis,
individual probe/target interactions are completely independent.
Since location of signal is determined by the length of the homologous
region of probe with target, several RNA targets and appropriate
controls may be assayed simultaneously.
Quantitation: Absolute
quantitation of an RNA species is straightforward and, as with
Northern analysis, involves generating a concentration curve of
synthetic sense strand target to which experimental sample signals
can be compared (see Ambion's Technical Bulletin #165).
While using probes from species divergent from the RNA sample can
be problematic (mismatches will be cleaved), cross-hybridization
(e.g. to multigene family sequences) a frustrating problem with
Northern analysis, is eliminated with RPAs.
Cons
Lack of Size Information: The
primary limitation of RPAs is that they do not reveal information
about message size. Protected fragment size is determined by the
length of the homologous region of probe with target - usually
only 200-400 nucleotides.
Limited Probe Choice: Another
drawback to RPAs is that only antisense RNA probes can be used.
In addition, the probe sequence must typically be completely homologous
to the target (except for a small stretch of vector sequence at
one or both ends of the probe). Therefore, partially related sequences
(e.g., cross species or gene families) usually cannot be analyzed.
RT-PCR
Reverse Transcription, coupled with the Polymerase
Chain Reaction (RT-PCR), has literally revolutionized the study
of gene expression. It is now possible to detect the RNA transcript
of any gene, regardless of the amount of starting material or the
relative abundance of the specific mRNA. In RT-PCR, an RNA template
is copied into a complementary DNA transcript (a cDNA) using a
retroviral reverse transcriptase. The cDNA sequence of interest
is then amplified exponentially using PCR. Detection of the PCR
product is typically performed by agarose gel electrophoresis and
ethidium bromide staining or by the use of radiolabeled nucleotides
or primers in the PCR.
Pros
Sensitivity: RT-PCR
is the most sensitive technique for mRNA detection and quantitation
currently available. Theoretically, a single copy of a message
can be detected by this technique. In practice, tens to hundreds
of copies are required for reliable quantitation.
Sample Integrity Requirements: Since
most RT-PCR methods amplify only a few hundred bases rather than
the complete mRNA sequence, the sample RNA can be slightly degraded.
Quantitation: Like
other methods of mRNA analysis, RT-PCR can be used for relative
or absolute quantitation. Relative quantitation compares transcript
abundance across multiple samples, using a co-amplified internal
control, which ideally has invariant expression within those samples,
for sample normalization. Absolute quantitation using competitive
RT-PCR measures the absolute amount of a specific mRNA sequence
in a sample. Dilutions of a synthetic RNA (identical in sequence,
but slightly shorter than the endogenous target) are added to sample
RNA replicates and are co-amplified with the endogenous target.
The PCR product from the endogenous transcript is then compared
to the concentration curve created by the synthetic "competitor
RNA." It is also possible to do real-time RT-PCR quantitation by
measuring an internal control in replicate samples.
Cons
Sample Purity Requirements: Because
of its sensitivity, the technique of RT-PCR requires that samples
be free of genomic DNA or other DNA contaminants. Special care
must be taken during RNA isolation to ensure that the sample RNA
is DNA-free. For more information, see Technical Bulletins #176 and
#181.
Optimization Requirements: RT-PCR
can be the most technically challenging RNA quantitation method
of those discussed here. It often requires substantial pre-experimental
planning to design suitable primers and controls.
In relative RT-PCR, the choice of internal standard
is critical. An ideal internal standard is one with invariant expression
during the cell cycle, between cell types, or in response to the
experimental treatment under analysis. Also, in relative RT-PCR,
the products must be analyzed while the PCR is still in exponential
phase for both the target and the reference amplicon. Thus pilot
experiments are required both to validate the internal control
and to determine cycling parameters for the exponential amplification
phase of all targets to be studied. For more information, see RT-PCR:
The Basics and Technical Bulletin #151.
Competitive RT-PCR makes use of an exogenous
RNA transcript (competitor) that must be accurately quantitated
and added to replicate samples in amounts that span the range of
the target mRNA levels. Experimentation is needed to determine
the amount of competitor required and to ensure that the target
and competitor sequences are amplified with equivalent efficiencies
yet are discernible by gel electrophoresis. For information on
designing and validating competitors, see Technical Bulletin #185.
Clearly, if multiple samples will be analyzed, competitive RT-PCR
becomes technically laborious and costly since multiple RT and
PCR reactions are required for each sample.
Summary
Despite its limitations, RT-PCR is currently
the accepted approach for quantitation of extremely rare transcripts
from minute samples. However, for targets within the limit of detection
of Northern and RPA analysis, these techniques are preferred because
of the linearity and simplicity of these assays. Incremental improvements
in the sensitivity of Northern and RPA analysis steadily increase
the utility of these techniques and broaden their appeal.
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