Gene Expression Detection Assay for Cancer Clinical Use

Narrandes, Shavira; Xu, Wayne

The specificity for each of the qRT-PCR, DNA microarray, NanoString nCounter, MiSeq RNA-Seq, and TMA depends on the primers/probes and/or reagents being used. As previously discussed, a strict design is required to ensure the qRT-PCR primer tightly anneals to the mRNA transcript and amplifies only the section of interest. Therefore, the specificity of qRT-PCR depends on the forward and reverse primer design, as well as the oligonucleotide probe used in detection. 11, 12, 97 The specificity of DNA microarrays depends on a number of factors, such as the density of probes annealed to the slide, the single probe (hybridization of a probe to a single target), single spot (a spot of multiple probes may contain probes that are perfectly or partially hybridized to a target), and spot-set (multiple spots represent different sections of the same sequence). 98 Similar to PCR, the specificity may also depend on the probe design; for example, cDNA microarrays can employ probes up to a few thousand base pairs in length while oligonucleotide arrays perform best with shorter probes that can be 25-30mer or 60-70mer in length. 83 The specificity of NanoString nCounter is due to its target-specific Capture and Reporter probes, as these are designed to anneal to a nucleic acid region of interest. These combine with the internal controls used to form the CodeSet, which confers the overall specificity. 99 TMA quality is dependent on the type of analysis being done. For example, FISH, RNA FISH, and immunohistochemistry each confer different specificities to the TMA assay. 100 , 101 RNA probes about 250-1500 nucleotides in length, with a common recommendation of 800 nucleotides, tend to demonstrate high specificity. 102 Chemicallysynthesized oligonucleotides labelled with fluorophores and calibrated to a certain region tend to generate RNA FISH probes with high specificity. 100