Integrity assay for messenger RNA in mouse and human brain samples and synaptosomal preparations.

Autor:	Bujanauskiene D; VU LSC-EMBL Partnership Institute for Genome Editing Technologies, Life Sciences Center, Vilnius University, Vilnius, Lithuania.; Institute of Biosciences, Life Sciences Center, Vilnius University, Vilnius, Lithuania., Merkevicius K; Institute of Biosciences, Life Sciences Center, Vilnius University, Vilnius, Lithuania.; Clinic of Pediatrics, Institute of Clinical Medicine, Faculty of Medicine, Vilnius University, Vilnius, Lithuania., Kuliesiute U; VU LSC-EMBL Partnership Institute for Genome Editing Technologies, Life Sciences Center, Vilnius University, Vilnius, Lithuania.; Institute of Biosciences, Life Sciences Center, Vilnius University, Vilnius, Lithuania., Denkovskij J; Department of Regenerative Medicine, Center for Innovative Medicine, Vilnius, Lithuania., Kutanovas S; VU LSC-EMBL Partnership Institute for Genome Editing Technologies, Life Sciences Center, Vilnius University, Vilnius, Lithuania., Luksys G; Center of Neurosurgery, Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania.; Clinic of Neurology and Neurosurgery, Faculty of Medicine, Vilnius University, Vilnius, Lithuania., Rocka S; Center of Neurosurgery, Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania.; Clinic of Neurology and Neurosurgery, Faculty of Medicine, Vilnius University, Vilnius, Lithuania., Bernotiene E; Department of Regenerative Medicine, Center for Innovative Medicine, Vilnius, Lithuania., Neniskyte U; VU LSC-EMBL Partnership Institute for Genome Editing Technologies, Life Sciences Center, Vilnius University, Vilnius, Lithuania.; Institute of Biosciences, Life Sciences Center, Vilnius University, Vilnius, Lithuania.
Jazyk:	angličtina
Zdroj:	IScience [iScience] 2024 Jun 29; Vol. 27 (8), pp. 110419. Date of Electronic Publication: 2024 Jun 29 (Print Publication: 2024).
DOI:	10.1016/j.isci.2024.110419
Abstrakt:	Traditionally, RNA integrity evaluation is based on ribosomal RNAs (rRNAs). Nevertheless, gene expression studies are usually focused on protein-coding messenger RNAs (mRNAs). Here, we present an RT-qPCR-based assay, which estimates mRNA integrity by comparing the abundance of 3' and 5' mRNA fragments. The assay was validated using plasmids with cloned 3'- and 5'-ends of the cDNA reflecting different ratios of 3' and 5' cDNA amplicons in partially degraded RNA samples. The accuracy of integrity value was ensured by including primer efficiency. We used 5':3' assay to quantify RNA degradation in heat- and enzyme-degraded mouse and human brain tissue RNA as well as in clinical human brain RNA samples. In addition, the 5':3' assay was suitable for assessing mRNA integrity in synaptosomal preparations that lack rRNAs. We concluded that the 5':3' assay can be used as a reliable method to evaluate mRNA integrity in tissue and subcellular preparations. Competing Interests: The authors declare no competing interest. (© 2024 The Authors.)
Databáze:	MEDLINE
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