Isoformenquantifizierung

Isoform quantification (German: Isoformenquantifizierung) is the measurement of expression levels of alternative isoforms of transcripts or proteins derived from the same gene. Isoforms arise primarily through alternative splicing, alternative promoter usage, and alternative polyadenylation, leading to distinct mRNA transcripts and, in many cases, distinct protein products. Quantifying isoforms enables researchers to study regulatory mechanisms, tissue specificity, and functional diversity, as well as disease-associated changes in isoform usage.

In transcriptomics, isoform quantification is performed by sequencing RNA (RNA-seq) and estimating transcript abundance. Reads may map to multiple isoforms, so statistical methods estimate isoform-level counts. Popular tools include RSEM, Kallisto, Salmon, and StringTie. Outputs commonly include transcripts per million (TPM), fragments per kilobase of transcript per million mapped reads (FPKM or RPKM), or raw counts. High-quality isoform quantification relies on comprehensive transcript annotations and, increasingly, long-read sequencing (PacBio Iso-Seq, Oxford Nanopore) to define isoform structures more accurately.

Protein isoform quantification uses mass spectrometry-based proteomics. Quantification relies on distinguishing peptides unique to specific isoforms; shared peptides complicate attribution. Approaches include label-free quantification, spectral counting, and isobaric tagging methods such as TMT or iTRAQ. Outputs report relative abundances of isoforms or proteins, requiring robust protein annotations and careful computation to avoid misassignment.

Differential isoform expression analysis compares isoform abundances across conditions to identify isoform switches and regulatory events with functional consequences. Isoform quantification is essential for understanding gene function, biomarker discovery, and disease mechanisms, but faces challenges including incomplete annotations, multi-mapping reads, and detection bias in proteomics. Ongoing improvements in sequencing technologies and analytic methods continue to enhance isoform-resolution studies.