A first step in working out how a gene functions is to track its expression at the transcript or protein levels. Different starting sources can be used and different technologies can be employed with varying resolution and throughput (see Table 1).
Table1. DIFFERENT LEVELS OF EXPRESSION MAPPING
Crude RNA/cDNA or protein extracts are frequently used as source material. Sometimes, however, expression is sampled in tissue sections or even whole embryos that have been fixed so as to preserve the original in vivo morphology. Gene expression can also be studied in live cells in tissue culture, and even in certain living experimental organisms that have optically transparent tissues at certain stages in development (allowing tracking of expression of genes tagged with a fluorescent chemical group).
Laser capture microdissection uses a laser to dissect out microscopic portions of a tissue to produce pure cell populations from sources such as tissue biopsies and stained tissue, and even single cells. As a result, gene expression analyses can be focused on single cells or on homogeneous cell populations that will be more representative of the in vivo state than cell lines.
Low-resolution expression patterns are initially sought from genes by tracking gross expression in RNA extracts or protein extracts. In addition to being able to sample expression in different tissues, these patterns may provide useful information on the level of expression and on expression-product variants that can differ in size (isoforms). Interesting expression patterns can be followed-up using methods to track expression within a cell, or within groups of cells and tissues that are spatially organized in a manner representative of the normal in vivo organization.
Low-throughput expression screening follows the expression of only one or a very small number of genes at a time. High-throughput methods can simultaneously track the expression of very many, often thousands of genes at a time, and can offer whole genome expression screening.
Quantifying the expression of individual genes using real-time (quantitative) PCR
Various PCR-based methods can track gene expression in cell types or tissues that are not easy to access in great quantity. To do that, the starting RNA is first copied into a complementary DNA sequence. In reverse transcriptase-PCR (RT-PCR), a cDNA copy is made of RNA using an oligo(dT) primer or random sequence oligonucleotide primers, and is then used to initiate a PCR. After the PCR is completed, an aliquot is submitted to standard gel electrophoresis. The size-fractionated DNA is exposed to a DNA-binding chemical such as ethidium bromide that allows the DNA to be visualized under ultraviolet light.
The basic RT-PCR method has been useful for identifying and studying different RNA isoforms of RNA transcript but is not suited to quantification (detection of DNA by ethidium bromide fluorescence is not very sensitive; amplicons have gone through a full 25–30 PCR cycles but, by this stage, the exponential stage of amplification has long since passed—see Figure 1 for the stages of a PCR assay). To obtain accurate quantification of RNA, a kinetic reaction, quantitative PCR (qPCR), is used: while the PCR is progressing, the amplification products are continuously quantified in a specialized PCR machine. To get the most accurate data, the measurements are made only during the early stages in the PCR assay, when the amplification is still exponential.
Fig1. Different phases in a polymerase chain reaction (PCR). After a lag phase, the amount of PCR product increases gradually at first. In the exponential phase, beginning after about 16–18 cycles and continuing to approximately the 25th cycle, the amount of PCR product is taken to be proportional to the amount of input DNA; quantitative PCR measurements are made on this basis. With further cycles, the amount of product increases at first but then tails off as the saturation phase approaches, when the reaction efficiency diminishes as reaction products increasingly compete with the remaining primer molecules for template DNA.
In order to detect amplification products during a qPCR assay, some measurable signal must be generated that is proportional to the amount of the amplified product. Current detection methods use fluorescence technologies, and the detection method may be nonspecific or specific (see Box 7.5).
High-resolution expression mapping by in situ hybridization and immunocytochemistry
High-resolution spatial expression patterns of RNA in tissues and groups of cells are normally obtained by tissue in situ hybridization or whole-embryo in situ hybridization (embryonic tissues are often used because their miniature size allows screening of many tissues in a single section). In tissue in situ hybridization, tissues are frozen or embedded in wax, then sliced using a microtome to give very thin sections (often 5 μm or less) that are mounted on a microscope slide. Hybridization of a suitable gene-specific probe to the tissue on the slide can then give detailed expression images representative of the distribution of the RNA in the tissue of origin (Figure 2).
Fig2. High-resolution gene expression studies using in situ hybridization and immunocytochemistry. (A) Principle of generating antisense riboprobes. A coding DNA sequence from a gene of interest is cloned in reverse orientation into a multiple cloning site (MCS) of a plasmid expression vector that has the powerful SP6 phage promoter (SP6 Pro) adjacent to the MCS. The phage RNA polymerase is used to transcribe the inverted gene sequence in the presence of ribonucleotide precursors, one of which has a labeled group (filled red circle) that gets incorporated into the antisense RNA transcripts. (B) In situ hybridization. The example shows expression of the Fgf8 fibroblast growth factor gene in a chick embryo at Hamburger Hamilton stage 20 (about 3 days of embryonic development after egg laying). Transcripts were labeled with an antisense digoxigenin-labeled Fgf8 probe and were detected with antidigoxigenin antibodies coupled to alkaline phosphatase. The alkaline phosphatase assay used a combination of BCIP (5-bromo-4-chloro-3-indolyl-phosphate) and NBT (nitro blue tetrazolium), resulting in deep-blue expression signals. Expression was evident in the developing eye, isthmus, branchial arches, somites, limb buds, and tailbud. (C) Immunocytochemistry. In this example, β-tubulin expression was screened in a transverse section of the brain of a 12.5-day mouse embryo. The antibody detection system used identifies β-tubulin expression ultimately as a brown color reaction (based on horseradish peroxidase/3,3′ diaminobenzidine). The underlying histology was revealed by counterstaining with a toluidine blue stain. LV, lateral ventricle; D, diencephalon; P, pons. (B, image kindly provided by Dr Terence Gordon Smith, Newcastle University; C, courtesy of Steve Lisgo, Newcastle University.)
Using suitably labeled probes, specific RNA sequences can also be tracked within single cells to identify sites of RNA processing, transport, and cytoplasmic localization. By using quantitative fluorescence in situ hybridization (FISH) and digital imaging microscopy, it has even been possible to visualize single RNA transcripts in situ. A further refinement uses combinations of different types of oligonucleotide probe labeled with spectrally distinct fluorophores. This has allowed transcripts from multiple genes to be tracked simultaneously.
Immunocytochemistry
Because of their exquisite diversity, selectivity, and sensitivity in detecting proteins, antibodies are ideally placed to track gene expression at the protein level. Specific anti bodies are prepared, labeled in some way, and then allowed to bind to proteins in cells and tissues, after which the bound label is detected.
Antibodies can be labeled in different ways. In direct detection methods, the purified antibody is labeled by attaching a reporter molecule, often a fluorophore or biotin, allowing the labeled antibody to bind directly to the target protein. Alternatively, the target protein is first bound by an unlabeled primary antibody that binds to the target protein, and this antibody is then specifically bound in turn by a suitably labeled secondary molecule that may be a secondary antibody, a specific antibody raised against the primary antibody. Sometimes a general secondary molecule is used, such as protein A, a protein found in the cell wall of Staphylococcus aureus. (Protein A happens to bind strongly to a common core region on antibody molecules—in the second and third constant regions of the Fc portion of immunoglobulin heavy chains.)
In immunocytochemistry (also referred to as immunohistochemistry) an antibody is used to obtain an overall expression pattern for a protein within a tissue or other multi cellular structure. As in tissue in situ hybridization, the tissues are typically either frozen or embedded in wax and then cut into very thin sections with a microtome before being mounted on a slide. A suitably specific antibody is allowed to bind to the protein in the tissue section and can produce expression data that can be related to histological staining of neighboring tissue sections (see Figure 1C).
