Figure 8–62 In situ hybridization to mRNAs has been used to generate an atlas of gene expression in the mouse brain. This computer-generated image shows the expression of several different mRNAs specific to an area of the brain associated with learning and memory. Similar maps of expression patterns of all known genes in the mouse brain are compiled in the brain atlas project, which is available online. (From M. Hawrylycz et al., PLoS Comput. Biol. 7:e1001065, 2011.)
Figure 8–63 RNA levels can be measured by quantitative RT-PCR. The fluorescence measured is generated by a dye that fluoresces only when bound to the double-stranded DNA products of the RT-PCR (see Figure 8–36). The red sample has a higher concentration of the mRNA being measured than does the blue sample, since it requires fewer PCR cycles to reach the same half-maximal concentration of double-stranded DNA. Based on this difference, the relative amounts of the mRNA in the two samples can be precisely determined.
that are expressed at reasonably high levels, and it is difficult to quantify (or even identify) rare RNAs. A more accurate method is based on the principles of PCR (Figure 8–63). Called quantitative RT-PCR (reverse transcription–polymerase chain reaction), this method begins with the total population of RNA molecules purified from a tissue or a cell culture. It is important that no DNA be present in the preparation; it must be purified away or enzymatically degraded. Two DNA primers that specifically match the mRNA of interest are added, along with reverse transcriptase, DNA polymerase, and the four deoxyribonucleoside triphosphates needed for DNA synthesis. The first round of synthesis is the reverse transcription of the RNA into DNA using one of the primers. Next, a series of heating and cooling cycles allows the amplification of that DNA strand by PCR (see Figure 8–36). The quantitative part of this method relies on a direct relationship between the rate at which the PCR product is generated and the original concentration of the mRNA species of interest. By adding chemical dyes to the PCR that fluoresce only when bound to double-stranded DNA, a simple fluorescence measurement can be used to track the progress of the reaction and thereby accurately deduce the starting concentration of the mRNA that is amplified. Although it seems complicated, this quantitative RT-PCR technique is relatively fast and simple to perform in the laboratory; it is currently the method of choice for accurately quantifying mRNA levels from any given gene.
Analysis of mRNAs by Microarray or RNA-seq Provides a Snapshot of Gene Expression
As discussed in Chapter 7, a cell expresses only a subset of the many thousands of genes available in its genome; moreover, this subset differs from one cell type to another or, in the same cell, from one environment to the next. One way to determine which genes are being expressed by a population of cells or a tissue is to analyze which mRNAs are being produced.
The first tool that allowed investigators to analyze simultaneously the thousands of different RNAs produced by cells or tissues was the DNA microarray. Developed in the 1990s, DNA microarrays are glass microscope slides that contain hundreds of thousands of DNA fragments, each of which serves as a probe for the mRNA produced by a specific gene. Such microarrays allow investigators to monitor the expression of every gene in a genome in a single experiment. To do the analysis, mRNAs are extracted from cells or tissues and converted to cDNAs (see Figure 8–31). The cDNAs are fluorescently labeled and allowed to hybridize to the fragments bound to the microarray. An automated fluorescence microscope then determines which mRNAs were present in the original sample based on the array positions to which the cDNAs are bound (Figure 8–64).