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.
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.
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.
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.
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. 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.
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.
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.
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. 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.