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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?

How do scientists measure gene expression?

A photograph shows a short-haired Siamese cat sitting against a white background. The cat has cream-colored fur on its head, neck, chest, back, abdomen, and hindquarters. Its tail, ears, face, front paws, and hind paws are dark brown. The left front paw is raised off the floor.
Figure 1: Siamese cats have colored \"points\" because of a temperature-sensitive pigmentation gene. In cooler areas of a cat's body (nose and paws), this gene is expressed to a greater degree.
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.

Both Northern blots and SAGE analyses work by measuring levels of mRNA, the intermediary between DNA and protein. Remember, in order to activate a gene, a cell must first copy the DNA sequence of that gene into a piece of mRNA known as a transcript. Thus, by determining which mRNA transcripts are present in a cell, scientists can determine which genes are expressed in that cell at different stages of development and under different environmental conditions.

Northern blots: What are they, and how do they work?

A photograph shows the results of three Northern blot analyses: one performed 15 minutes after exposure to a drug, one performed 1 hour after exposure, and one performed 2 hours after exposure. The three analyses are arranged side-by-side for comparison. The first lane in each blot contains a standard sample, with black bands marking specific RNA sizes. The second lane in each blot shows the RNA content for the treated sample. A thick black band appears halfway down the length of each blot. The band in the 15-minute analysis looks like a small circle, and is indicated by a red arrow. The band in the 1-hour analysis is a larger black oval, approximately twice the size as the band in the previous analysis. The mark in the 2-hour analysis is the largest black oval, and is close in size to the mark in the 1-hour analysis. It is indicated with a blue arrow.
Figure 2: A Northern blot allows comparison of mRNA levels, which are a direct reflection of gene expression. This blot above shows the tracking of mRNA levels in a cell sample at various intervals after exposure to a drug. At 15 minutes after exposure, mRNA levels are low (red arrow, small black mark); at two hours after exposure, these levels have increased (blue arrow, larger black mark).

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).

Is it possible to study the expression of multiple genes simultaneously?

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.

How does SAGE work?

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.

Capturing mRNA

To begin a SAGE analysis, researchers must first separate the mRNA in a sample from the other cellular contents. To do this, they attach long strips of thymine nucleotides to tiny magnetic beads. When researchers flush the contents of a cell over the beads, these thymine strips form complementary base pairs with the poly-A tails of the mRNA molecules. Thus, when the flushing process is complete, the mRNA transcripts from the sample are captured because they are attached to the magnetic beads, while the other contents of the cells flush past the beads and are discarded.

Rewriting mRNA into cDNA

A schematic shows 16 nucleotides arranged to form a single horizontal strand of MRNA. Below the single strand, 13 complementary nucleotides on a strand of CDNA are paired with the nucleotides on the upper strand. A 14th nucleotide is approaching the second strand. On both strands, gray horizontal cylinders represent the phosphate-sugar molecules, and vertical rectangles represent the nitrogenous bases. The nitrogenous bases on the top strand are green, blue, orange, or yellow, representing different chemical identities of the bases. In the bottom strand, the yellow bases have been replaced by red bases, indicating the switch from uracil in MRNA to thymidine in CDNA. A red circle below each of the phosphate-sugar molecules on the CDNA strand indicates that this strand has been fluorescently labeled.
Figure 3: Reverse transcription converts mRNA into cDNA.
mRNA is more fragile than DNA, which makes it difficult to handle and analyze. To solve this problem, researchers often convert mRNA samples into complementary DNA sequences, or cDNA. This is done by reversing the natural process a cell uses to make mRNA from DNA, a method known as reverse transcription. The reverse transcription process doesn't use DNA polymerase or RNA polymerase; instead, it employs a special enzyme called reverse transcriptase. This enzyme makes cDNA sequences that are complementary to each mRNA transcript, essentially creating a converted form of the same sequence (Figure 3). This new single-stranded cDNA is then converted into a double-stranded cDNA molecule.

Cutting tags from each cDNA

To begin the next portion of SAGE, the researchers use a cutting enzyme to slice off short segments of nucleotides, called tags, at designated positions in each cDNA molecule. Next, two tags from each cDNA are combined into a single unit. These tags then become the representative for the gene they came from, and they act as a unique identifier in the form of a stand-in. Without having to process the entire mRNA sequence thereafter, scientists can use these shorter tag sequences to keep track of whether a specific gene was expressed in mRNA form.

Linking tags together in chains for sequencing

After the different tags have been made from each mRNA sequence, they are next linked together into long chains called concatemers. These concatemers therefore contain representatives of mRNAs from a group of genes. Linking the tags together in a concatemer is important, because it means that researchers will later be able to read thousands of tags at once during the analysis portion of the SAGE procedure.

Copying and reading the chains

Although the researchers now possess concatemers representing the genes expressed in the sample, they need multiple copies of these concatemers if they wish to run the molecules through a sequencing machine. Thus, just before sequencing, the concatemers are inserted into bacteria, and through their own replication process these bacteria make millions of copies of each concatemer chain. This step increases the volume of material, and it therefore ensures that there is a baseline amount of material necessary for a sequencing machine. After that, researchers use a sequencing machine to decode and read the long string of nucleotides in each chain.

Identifying and counting the tags

Finally, a computer processes the data from the sequencing machine and compiles a list of tags. By comparing the tags to a sequence database, the researchers can identify the mRNA (and ultimately the gene) that each tag came from. By subsequently counting the number of times each tag is observed, the researchers can also estimate the degree to which a particular gene is expressed: the more often a tag appears, the greater the level of gene expression.

What can researchers learn from SAGE?

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.

Are there other ways to measure gene expression?

More about measuring gene expression

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.

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