Gene Expression Detection Assay for Cancer Clinical Use

Narrandes, Shavira; Xu, Wayne

High throughput refers to the advantage of a technology to simultaneously analyze many (i.e. hundreds of thousands) samples under a certain set of conditions. It therefore may depend on the number of genes that can be detected by a technique and/or the number of samples that can be processed. Well-plates that can hold a maximum of 384 samples can be purchased for PCR. Although control samples (tests priming the housekeeping genes) and all replicates (a recommended number of three) should be run on a single plate, a qRT-PCR assay has the potential to assess 384 samples at once. This substantial number of wells also allow for the analyses of a few hundred genes in Singleplex reactions. 12, 103 The number of samples analyzed on a DNA microarray can depend on the purpose of the microarray and the number of genes being assessed. Whole-genome arrays can be performed for an individual or a small number of individuals can be simultaneously analyzed for a certain set of genes on one array. For example, Prosigna is able to assess up to 8 breast cancer patient samples for 1900 features on a single array. 104 , 105 It has been reported that up to 50 000 different probes are available for detections on a microarray, with each detection representing a different gene. 106 The nCounter has the capacity to input 12 samples and detect 800 genes. 107 Approximately 1000 genes can be targeted and 96 samples can be processed using the MiSeq benchtop sequencer; clusters of about 1000 molecules or 1536 amplicons can be generated from a single transcript and anywhere from 700 to thousands of targets have been reported. 108 , 109 Similar to DNA microarrays, the number of samples assessed and genes detected on the TMA depends on its purpose. Generally, 1000 spots representing different samples can be arrayed on a TMA. 67, 68 Although many genes have the ability to be detected as different coloured probes can be designed to anneal to their target region, studies tend to depict 1-3 genes using fluorescent detection on a TMA. 74, 75, 76, 77 This may be because resolution can be difficult using many different probes in an in situ detection assay.