Fiche publication
Date publication
octobre 2024
Journal
Nucleic acids research
Auteurs
Membres identifiés du Cancéropôle Est :
Pr MOTORINE Iouri
,
Dr MARCHAND Virginie
Tous les auteurs :
Kristen M, Lander M, Kilz LM, Gleue L, Jörg M, Bregeon D, Hamdane D, Marchand V, Motorin Y, Friedland K, Helm M
Lien Pubmed
Résumé
Due to its high modification content tRNAs are notoriously hard to quantify by reverse transcription and RNAseq. Bypassing numerous biases resulting from concatenation of enzymatic treatments, we here report a hybrid approach that harnesses the advantages of hybridization-based and deep sequencing-based approaches. The method renders obsolete any RNAseq related workarounds and correction factors that affect accuracy, sensitivity, and turnaround time. Rather than by reverse transcription, quantitative information on the isoacceptor composition of a tRNA pool is transferred to a cDNA mixture in a single step procedure, thereby omitting all enzymatic conversations except for the subsequent barcoding PCR. As a result, a detailed tRNA composition matrix can be obtained from femtomolar amounts of total tRNA. The method is fast, low in cost, and its bioinformatic data workup surprisingly simple. These properties make the approach amenable to high-throughput investigations including clinical samples, as we have demonstrated by application to a collection of variegated biological questions, each answered with novel findings. These include tRNA pool quantification of polysome-bound tRNA, of tRNA modification knockout strains under stress conditions, and of Alzheimer patients' brain tissues.
Mots clés
RNA, Transfer, genetics, High-Throughput Nucleotide Sequencing, methods, Humans, DNA, Complementary, genetics, Nucleic Acid Hybridization, methods, Alzheimer Disease, genetics, Sequence Analysis, RNA, methods, Brain, metabolism
Référence
Nucleic Acids Res. 2024 10 14;52(18):e89
