Microarrays are a method of determining transcriptional activity, by hybridising the transcriptome of a cell to a glass slide. The glass slide has DNA probes with known sequences bound in spots of identical probes, and the mRNA transcripts are fluorescently labelled. On hybridising the mRNA to the DNA probes, they become immobilised. This allows fluorescence to be detected from each spot on the slide, which can then be converted to relative transcriptional activity.
An issue with microarrays for transcriptome activity can be with highly expressed genes. The fluorescence can overwhelm the reader, or there may be an excess of mRNA transcripts. This results in an underestimate of the transcriptional activity for that gene, which can cause issues with the relative transcription of other genes of interest.
Microarrays can only be used with smaller genomes, due to the limited size of the slides used. This means it cannot be used with Homo sapiens cells, for instance, due to the large transcriptome.
To overcome these two issues, of underestimating the relative transcription level, and large genomes, RNA-seq can be used. This is a technique using DNA sequencing. With Illumina sequencing (second generation sequencing), mRNA transcripts must first be converted to cDNA and then sequenced. Third generation sequencing does not require reverse transcription. The transcripts can then be pieced together into a consensus sequence (where they are longer than the read length of the sequencing technology, less of an issue with third generation sequencing), and the number of each transcript can be determined. This allows the identification of transcriptional levels in a more quantitative manner than microarrays.