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