figure 21.6. Polymerase chain reaction (PCR).
degraded, or otherwise unsatisfactory specimens. It is thus ideally suited to the small biopsies, cytology specimens, body fluids, or even laser-captured cells that are commonly used for nucleic acid assays. With suitable amplification (e.g., numbers of cycles), it is possible to detect the RNA from even single cells. A typical result for PCR amplification of MYCN in neuroblastoma after varying numbers of rounds of amplification is illustrated in Figure 21.6. Note that in this example, the MYCN-amplified and overexpressing specimen (from a needle biopsy) shows detectable product long before the single-copy control, indicating high-level expression. Despite the difference, this method is generally not used for absolute quantitation. Instead, quantitative real-time PCR has become the method of choice.
b. Quantitative PCR: Quantitative real-time PCR has become a standard laboratory diagnostic technology by virtue of its extraordinary sensitivity, amenability to quality control and reproducibility, speed, and cost. Although the product amplified is DNA, this is generally a cDNA produced by reverse transcriptase of mRNA. At least three manufacturers (e.g., Roche, ABI, and Cepheid) make devices and numerous reagent suppliers make various chemistries for DNA detection, including TaqMan (Roche, Basel, Switzerland) and Molecular Beacons probes, Amplifluor (Chemicon, Temecula, CA) and Scorpion primers (DxS, Ltd., Manchester, UK), and intercalating dyes such as SYBR Green (Finnzymes, Espoo, Finland). All have been used successfully. Regardless of the chemistry, in each case the point at which there is a detectable change in fluorescence intensity during the course of multiple cycles of DNA amplification is noted and termed the cycle threshold, Ct, the point termed the log-linear phase
figure 21.7. Quantitative real-time (QRT) PCR.
¡3—aclin figure 21.7. Quantitative real-time (QRT) PCR.
of amplification. This is a sensitive index of how much target is present in the sample, and is thus a highly reproducible measurement of the amount of RNA (or DNA) present in a sample. A typical result for detection of a chimeric oncogene in a Ewing's sarcoma (e.g., EWS-FLI1) is illustrated in Figure 21.7, where it is clear that the unknown sample shows a cycle threshold and subsequent amplification virtually indistinguishable from the positive control and quite distinct from the negative control.
By far the most dramatic advance in the detection of mRNA has been the development of microarray technology during the past decade. These arrays, whether constructed of cDNA, oligomeric DNA, or in situ synthesized 25-mer oligomeric DNA, share the ability to detect thousands of RNA transcripts simultaneously. From a few hundred "spots" a decade ago, the current generation of arrays routinely assays all known, and many unknown, genes from the entire transcrip-tome. For example, the current generation of Affymetrix
(Santa Clara, CA) GeneChips, the U133 plus 2, assesses the expression of 47,000 unique RNA transcripts, a number considerably larger than the estimated number of genes in the human genome. The identity of more than half is unknown. The major impact of these arrays has been their ability to simultaneously measure the gene expression activity of any and all genes, and deliver a quantitative value, resulting in a vast number of gene by gene measurements. These gene expression profiles are markedly different for different tissues and tumors and have been shown to provide powerful diagnostic information that in many cases is superior to any antecedent diagnostic technology, on a par with an expert pathologist, and far more reproducible.
a. cDNA: The first-generation microarrays were created by spotting cDNAs of specific sequence, complementary to known genes, usually by fine-tipped metal probes held in physical arrays. The arrays were then "stamped" with the same cDNA in the same position, one after another. The resultant arrays are then incubated with a dual sample: a mixture of control and tumor, for example, in balanced proportion, each with a different color fluor (typically Cy3 and Cy5). The result is either red, green, yellow, or black, or variations thereon. A typical result is illustrated in Figure 21.8a. Red or green denotes 100% hybridization by only one of the RNA moieties in the mix (e.g., control or tumor, for example); yellow indicates balanced competitive hybridization of both in about equal proportion; and black indicates no hybridization by either. By calculating the ratios of red to green, the "fold change," or multiples of greater or lesser expression of sample versus control, can be calculated.
Although this method is still in use, it has become considerably less popular with recognition that cDNA clones are prone to cross-hybridization, are often not monoclonal, and may in fact be mislabeled. This intrinsic lack of control over the actual sequence present on the array has prompted the search for more precise nucleotide probes for arrays.
b. Spotted oligomeric DNA: In an effort to improve the reproducibility of spotted arrays, most laboratories are increasingly using defined oligomers (usually 50-mers or 60-mers) of known sequence. Advantages of this approach include the ability to precisely replicate arrays from newly synthesized oligomers and achieve the same results, the
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Among the evils which a vitiated appetite has fastened upon mankind, those that arise from the use of Tobacco hold a prominent place, and call loudly for reform. We pity the poor Chinese, who stupifies body and mind with opium, and the wretched Hindoo, who is under a similar slavery to his favorite plant, the Betel but we present the humiliating spectacle of an enlightened and christian nation, wasting annually more than twenty-five millions of dollars, and destroying the health and the lives of thousands, by a practice not at all less degrading than that of the Chinese or Hindoo.