Molecular Biotechnology. Bernard R. Glick

or gene chip) experiment consists of hybridizing a nucleic acid sample (target) derived from the mRNAs of a cell or tissue to single-stranded DNA sequences (probes) that are arrayed on a solid platform. Depending on the purpose of the experiment, the probes on a microarray may represent an entire genome, a single chromosome, selected genomic regions, or selected coding regions from one or several different organisms. Some DNA microarrays contain sets of oligonucleotides as probes, usually representing thousands of different genes, that are synthesized directly on a solid surface. Thousands of copies of an oligonucleotide with the same specific nucleotide sequence are synthesized in a predefined position on the array surface (probe cell). The probes are typically 20 to 70 nucleotides, although longer probes can also be used, and several probes with different sequences for each gene are usually present on the microarray to minimize errors. Probes are designed to be specific for their target sequences, to avoid hybridization with nontarget sequences, and to have similar melting (annealing) temperatures so that all target sequences can bind to their complementary probe sequence under the same conditions. A complete whole-genome oligonucleotide array may contain more than 500,000 probes representing as many as 30,000 genes.

      For most gene expression profiling experiments that utilize microarrays, mRNA is extracted from cells or tissues and used as a template to synthesize cDNA using reverse transcriptase. Usually, mRNA is extracted from two or more sources for which expression profiles are compared, for example, from diseased versus normal tissue, or from cells grown under different conditions (Fig. 2.45A). The cDNA from each source is labeled with a different fluorophore by incorporating fluorescently labeled nucleotides during cDNA synthesis. For example, a green-emitting fluorescent dye (Cy3) may be used for the normal (reference) sample and a red-emitting fluorescent dye (Cy5) for the test sample. After labeling, the cDNA samples are mixed and hybridized to the same microarray (Fig. 2.45A). Replicate samples are independently prepared under the same conditions and hybridized to different microarrays. A laser scanner determines the intensities of Cy5 and Cy3 for each probe cell on a microarray. The ratio of red (Cy5) to green (Cy3) fluorescence intensity of a probe cell indicates the relative expression levels of the represented gene in the two samples (Fig. 2.45B). To avoid variation due to inherent and sequence-specific differences in labeling efficiencies between Cy3 and Cy5, reference and test samples are often reversed labeled and hybridized to another microarray. Alternatively, for some microarray platforms, the target sequences from reference and test samples are labeled with the same fluorescent dye and are hybridized to different microarrays. Methods to calibrate the data among microarrays in an experiment include using the fluorescence intensity of a gene that is not differentially expressed among different conditions as a reference point (i.e., a housekeeping gene), including spiked control sequences that are sufficiently different from the target sequences and therefore bind only to a corresponding control probe cell, and adjusting the total fluorescence intensities of all genes on each microarray to similar values under the assumption that a relatively small number of genes are expected to change among samples.

      Figure 2.45 Gene expression profiling with a DNA microarray. (A) mRNA is extracted from two samples (sample 1 and sample 2), and during reverse transcription, the first cDNA strands are labeled with the fluorescent dyes Cy3 and Cy5, respectively. The cDNA samples are mixed and hybridized to an ordered array of either gene sequences or gene-specific oligonucleotides. After the hybridization reaction, each probe cell is scanned for both fluorescent dyes and the separate emissions are recorded. Probe cells that produce only a green or red emission represent genes that are transcribed only in sample 1 or 2, respectively; yellow emissions indicate genes that are active in both samples; and the absence of emissions (black) represents genes that are not transcribed in either sample. (B) Fluorescence image of a DNA microarray hybridized with Cy3- and Cy5-labeled cDNA. Reproduced with permission from http://biotech.biology.arizona.edu/Resources/DNA_analysis.html. Courtesy of N. Anderson, University of Arizona.

      Genes whose expression changes in response to a particular biological condition are identified by comparing the fluorescence intensities for each gene, averaged among replicates, under two different conditions. The raw data of the fluorescence emissions of each gene are converted to a ratio, commonly expressed as fold change. Generally, positive ratios represent greater expression of the gene in the test sample than in the reference sample. Negative values indicate a lower level of expression in the test sample relative to the reference sample. The data are often organized into clusters of genes whose expression patterns are similar under different conditions or over a period of time (Fig. 2.46). This facilitates predictions of gene products that may function together in a pathway.

      Figure 2.46 Gene expression profile of cirrhotic liver tissue. Columns 1 to 7 and 8 to 15 are expression data from liver samples from patients with ethanol- and hepatitis virus C-induced cirrhosis of the liver, respectively. Each patient’s sample was compared to normal liver tissue. A total of 2,965 genes were differentially expressed. The asterisks denote patients with severe cirrhosis of the liver. Adapted from Figure 1 in Lederer, S. L., et al., Virol J . 3:98, 2006.

      The gene expression profile in Fig. 2.46 determined by microarray analysis clearly shows that different genes are transcribed in patients with cirrhosis of the liver compared to normal individuals, and in patients with ethanol-induced cirrhosis compared to those with cirrhosis induced by the hepatitis C virus. Moreover, there is a difference between the genes that are turned on during advanced ethanol-induced liver damage compared to those with less severe ethanol-induced cirrhosis (Fig. 2.46). No such distinction is evident among individuals with different severities of virus-induced cirrhosis (Fig. 2.46). In addition, information about the transcription of genes that contribute to a particular pathway or cellular activity can be extracted from a gene expression profile. For example, genes that are transcribed during lymphocyte proliferation and activation are highly expressed in viral-induced liver cirrhosis and to a much lesser extent in ethanol-associated cirrhotic samples (Fig. 2.47).

      Figure 2.47 Gene expression profile of lymphocyte-specific genes from cirrhotic liver tissue. Columns 1 to 7 and 8 to 15 are expression data from liver samples from the patients described in Fig. 2.46 with ethanol- and hepatitis virus C-induced cirrhosis of the liver, respectively. Each patient’s sample was compared to normal liver tissue. The cluster consists of about 70 genes. The asterisks denote patients with severe cirrhosis of the liver. Adapted from Figure 2B in Lederer, S. L., et al., Virol J. 3:98, 2006.

      RNA Sequencing

      Similar to microarrays, RNA sequencing is used to detect and quantify the complete set of gene transcripts produced by cells under a given set of conditions. In addition, RNA sequencing can delineate the beginning and end of genes, reveal posttranscriptional modifications such as variations in intron splicing that lead to variant proteins, and identify differences in the nucleotide sequence of a gene among samples. In contrast to microarray analysis, this approach does not require prior knowledge of the genome sequence, avoids high background due to nonspecific hybridization, and can accurately quantify highly expressed genes (i.e., probe saturation is not a concern as it is for DNA microarrays). Traditionally, RNA sequencing approaches required generating cDNA libraries from isolated RNA and sequencing the cloned