Quantifying subpercent nuclear <scp>TDP</scp> ‐43 loss in cells and <scp>ALS</scp> cortex using junction‐specific cryptic exon <scp>RT</scp> ‐ <scp>qPCR</scp>

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are progressive neurodegenerative diseases characterised by nuclear TDP-43 loss. Its hallmark, cryptic exon (CE) splicing, is often masked in bulk tissue analyses by the low abundance of affected neurons. We developed an ultrasensitive RT-qPCR assay targeting STMN2 CE using one exon-CE junction-spanning primer and the other within the CE. The design expands the dynamic range sevenfold: TDP-43 knockdown boosted STMN2 CE levels 1395-fold in differentiated SH-SY5Y neurons. Spike-in tests set detection at 0.16% deficient cells. Crucially, the assay revealed a 42-fold CE increase in ALS motor cortex, previously missed by conventional primers. This streamlined tool enables precise quantification of TDP-43 dysfunction and sensitive pharmacodynamic monitoring for future ALS-FTD therapeutic studies. Impact statement Because cryptic-exon signals are diluted in bulk tissue, we developed a junction-spanning STMN2 RT-qPCR with sub-percent sensitivity. This deployable biomarker will aid ALS/FTD researchers and drug developers by standardizing measurements and enabling sensitive pharmacodynamic monitoring of therapies targeting nuclear TDP-43 dysfunction.