| Title |
Integrity assay for messenger RNA in mouse and human brain samples and synaptosomal preparations / |
| Authors |
Bujanauskienė, Daina ; Merkevičius, Kajus ; Kuliešiūtė, Ugnė ; Denkovskij, Jaroslav ; Kutanovas, Simonas ; Lukšys, Gediminas ; Ročka, Saulius ; Bernotienė, Eiva ; Neniškytė, Urtė |
| DOI |
10.1016/j.isci.2024.110419 |
| Full Text |
|
| Is Part of |
iScience.. Elsevier Inc.. 2024, vol. 27, iss. 8, art. no. 110419, p. [1-16].. ISSN 2589-0042. eISSN 2589-0042 |
| Keywords [eng] |
Biochemistry methods ; Methodology in biological sciences ; Molecular biology |
| Abstract [eng] |
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. |
| Published |
Elsevier Inc |
| Type |
Journal article |
| Language |
English |
| Publication date |
2024 |
| CC license |
|
