Table 1.
Comparison of qRT-PCR, DNA Microarray, NanoString nCounter, Illumina MiSeq RNA-Seq, and Tissue Microarray assay properties.
| Assay | qRT-PCR | DNA Microarray | NanoString nCounter | Illumina MiSeq RNA-Seq | Tissue microarray & FISH |
|---|---|---|---|---|---|
| Primer/probe design | Gene-specific primer with attached quencher and reporter fluorophores; SYBR green | DNA oligo probes complementary to cDNA samples | Capture probe with 3' affinity tag and Reporter probe with colour-coded tag | Primers on flow cell and adaptors to ligate to ends of sample | Gene-specific RNA probes; gene-specific fluorochrome-labelled probes; monoclonal antibodies |
| Sample preparation | RNA extraction; reverse transcribe sample | RNA extraction; reverse transcribe sample, fragmentation | RNA extraction | RNA extraction; reverse transcribe sample; fragmentation, library construction | Map donor block; place into recipient block; make TMA |
| Instrument | Thermal cycler | Microarray scanner | Prep Station and Digital Analyzer | MiSeq benchtop sequencer | Tissue arrayer; microscope or array scanner |
| Reproducible | Yes | Yes | Yes | Yes | Yes |
| Specificity | Forward and reverse primer design, oligonucleotide probe | Density of probes annealed to the slide, probe design | Design of Capture and Reporter probes | Rely on data analysis | Rely on probes to be used |
| Sensitivity | 10-200 copies/cell | 1-10 copies/cell | <1 copy/cell | <1 copy/cell | 1-10 copies/cell |
| Clinic study | Yes | Yes | Yes | Yes | Yes |
| Commercialized for clinical use | Oncotype DX | MammaPrint | Prosigna | No | No |
| Number of genes or transcripts detected | 1-100 | 50 000 | 800 | Whole transcriptome | 3 |
| Up to sample# per assay | 1-96 | 1-12/array | 12 | 96 | 1000 |
| Processing steps | Prep reaction mixture, PCR cycles, Result analysis |
Label cDNAs, hybridization to array, Data analysis |
Label probes, hybridization to array, Data analysis |
cDNA lib prep, sequencing, data analysis |
Make TMA, Slide TMA Staining, Analysis |
| Raw Data analysis | by machine in 30 minutes | by machine in 1 hour 40 minutes | by machine in 2.7 hours | by machine in 3 hours | by machine or microscopy in 6 minutes |
| Normalization | 3-5 housekeeping genes | Housekeeping genes; RMA; LOWESS method | Housekeeping genes; positive controls | RPKM | Tissue array co-occurrence matric analysis |
| Data analysis | Absolute and relative quantification; standard dissociation curve; statistical tests | Visualization; statistical tests | Colour-coded images are taken and output as code counts | Data output as sequenced reads with quality scores or read alignments | PCR; H and E staining; FISH, ISH; fluorescent microscopy |