Gene Expression Is Analyzed by Tracking RNA

Even though nearly every cell in an organism's body contains the same set of genes, only a fraction of these genes are used in any given cell at any given time. It is this carefully controlled pattern of what is called "gene expression" that makes a liver cell different from a muscle cell, and a healthy cell different from a cancer cell. But how can researchers determine which genes are "turned on" and when?

Gene expression is dynamic, and the same gene may act in different ways under different circumstances. For example, imagine that two organisms have similar genotypes but different phenotypes. What is the cause of this variation in phenotype? Could the difference stem from differing regulation of gene expression? Could temperature affect expression of the organism's DNA (Figure 1)? Might some other factor be responsible? To go about answering these types of questions, researchers often use laboratory techniques such as a Northern blot or serial analysis of gene expression (SAGE). Both of these techniques make it possible to identify which genes are turned on and which are turned off within cells. Subsequently, this information can be used to help determine what circumstances trigger expression of various genes.

The quantity of mRNA transcript for a single gene directly reflects how much transcription of that gene has occurred. Tracking of that quantity will therefore indicate how vigorously a gene is transcribed, or expressed. To visualize differences in the quantity of mRNA produced by different groups of cells or at different times, researchers often use the method known as a Northern blot. For this method, researchers must first isolate mRNA from a biological sample by exposing the cells within it to a protease, which is an enzyme that breaks down cell membranes and releases the genetic material in the cells. Next, the mRNA is separated from the DNA, proteins, lipids, and other cellular contents. The different fragments of mRNA are then separated from one another via gel electrophoresis (a technique that separates molecules by passing an electrical current through a gel medium containing the molecules) and transferred to a filter or other solid support using a technique known as blotting. To identify the mRNA transcripts produced by a particular gene, the researchers next incubate the sample with a short piece of single-stranded RNA or DNA (also known as a probe) that is labeled with a radioactive molecule. Designed to be complementary to mRNA from the gene of interest, the probe will bind to this sequence. Later, when the filter is placed against X-ray film, the radioactivity in the probe will expose the film, thereby making marks on it. The intensity of the resulting marks, called bands, tells researchers how much mRNA was in the sample, which is a direct indicator of how strongly the gene of interest is expressed (Figure 2).

Until recently, scientists studied gene expression by looking at only one or very few gene transcripts at a time. Thankfully, new techniques now make large-scale studies of gene expression possible. One such technique is SAGE (serial analysis of gene expression). A method for measuring the expression patterns of many genes at once, SAGE not only allows scientists to analyze thousands of gene transcripts simultaneously, but it also enables them to determine which genes are active in different tissues or at different stages of cellular development.

SAGE identifies and counts the mRNA transcripts in a cell with the help of short snippets of the genetic code, called tags. These tags, which are a maximum of 14 nucleotides long, enable researchers to match an mRNA transcript with the specific gene that produced it. In most cases, each tag contains enough information to uniquely identify a transcript. The name "serial analysis" refers to the fact that tags are read sequentially as a continuous string of information.

The basic steps of the SAGE technique are outlined below.

Compared to other techniques for measuring gene expression, SAGE offers a significant advantage because it measures the expression of both known and unknown genes. Sometimes, when analyzing SAGE data, computers cannot find matches for certain tags in their sequence databases. What does this mean? Interestingly, a lack of matches indicates that the mRNA used to produce these tags is associated with genes that have not been studied before. In this way, SAGE has been used to discover new genes involved in a variety of diseases.

In addition to Northern blot tests and SAGE analyses, there are several other techniques for analyzing gene expression. Most of these techniques, including microarray analysis and reverse transcription polymerase chain reaction (RT-PCR), work by measuring mRNA levels. However, researchers can also analyze gene expression by directly measuring protein levels with a technique known as a Western blot.