Complementary DNA oligonucleotide direct in-gel quantification (cDINGQ) for precise tRNA fragment analysis.

tRNA-derived fragments have emerged as critical regulators in various biological processes, but reliable methods for their quantification remain a challenge due to their small size and extensive RNA modifications. In this study, we present the newly developed Complementary DNA Oligonucleotide Direct In-Gel Quantification (cDINGQ) method for tRF analysis and compare it with traditional radioactive [32P] northern blotting, nonradioactive approaches, and high-throughput Illumina sequencing under different experimental conditions. The cDINGQ method, utilizing Cy5-labeled hybridization probes, offers high specificity and sensitivity for detecting tRFs with significantly reduced processing time and costs. By applying these techniques to an Alzheimer's disease (AD) cell model, we demonstrate the reliability of these methods in detecting subtle variations in tRF abundance. Our findings highlight the sensitivity, specificity, and applicability of each method, addressing limitations such as RNA input requirements and probe hybridization conditions. The study further explores the utility of these methods for detecting tRFs in various biological contexts, emphasizing their potential for future research and biomarker discovery in disease-related studies.