Gene Expression Detection Assay for Cancer Clinical Use

Narrandes, Shavira; Xu, Wayne

qRT-PCR, DNA microarrays, NanoString nCounter, and Illumina MiSeq all require the isolation and purification of RNA. Following RNA extraction, qRT-PCR requires the mixing of various reagents before allowing the sample to reverse transcribe. A well-plate must be carefully prepared prior to subjecting the sample for the PCR and dissociation curve analysis steps in the thermal cycler machine and by the computer, respectively; this can take about 5 hours when including an RNA extraction step. Similarly, DNA microarrays require a reverse transcription step after the RNA extraction. Time is then required to allow the sample to hybridize to the array; the arrays can be pre-ordered and do not have to be constructed in the laboratory. The microarray scanner analyzes and slides and computer software can be purchased for data analysis, resulting in a total time of 20 hours. 126 NanoString nCounter only requires the user to prepare the initial sample and then transfer the sample to and from the Prep Station and Digital Analyzer machines. The machines carry out any processing and data analysis that needs to be done, resulting in a total of about 5.5 hours. 107 Similar to the nCounter, Illumina MiSeq only requires the user to prepare the library and load it onto the reagent cartridge; the rest of the sample processing and data analysis is carried out by the MiSeq benchtop sequencer, with the MiSeq Reporter analysis software launching following completion of the trial. A total of about 40 hours is required. 115 TMA construction is a tedious procedure and therefore requires the most work, demanding an average of up to two days, depending on the exact procedure followed. If donor tissue blocks are not available, one must collect samples prior to preparing the block. Then, slides of the tissue sample must be stained so that one can use the tissue arrayer to accurately remove cores from the donor block and place them on the slide. Following construction of the slide, reagents for FISH, ISH, or immunohistochemistry tests must be prepared and applied to the slide. The slides can then be analyzed by eye or with a (digital) slide scanner.