Identifying stable reference genes in polyethene glycol precipitated urinary extracellular vesicles for RT-qPCR-based gene expression studies in renal graft dysfunction patients

• Published in: Transplant immunology 2022-12, Vol.75, p.101715-101715, Article 101715
• Main Authors: Singh, Anula Divyash, Patnam, Sreekanth, Koyyada, Rajeswari, Samal, Rasmita, Alvi, Syed Baseeruddin, Satyanaryana, G., Andrews, Ravi, Panigrahi, Aswini Kumar, Rengan, Aravind Kumar, Mudigonda, Soma Sekhar, Maitra, Sanjay, Sasidhar, Manda Venkata
• Format: Article
• Language: English
• Subjects: Allograft dysfunction; Housekeeping genes (HK); Polyethylene glycol (PEG); Quantitative PCR (qPCR); RefFinder; Urinary biomarkers
• ISSN: 0966-3274; 1878-5492
• DOI: 10.1016/j.trim.2022.101715

Urinary extracellular vesicles (UEVs) hold RNA in their cargo and are potential sources of biomarkers for gene expression studies. The most used technique for gene-expression studies is quantitative polymerase chain reaction (qPCR). It is critical to use stable reference genes (RGs) as internal controls for normalising gene expression data, which aren't currently available for UEVs. UEVs were precipitated from urine of graft dysfunction patients and healthy controls by Polyethylene glycol, Mn6000 (PEG6K). Vesicular characterisation confirmed the presence of UEVs. Gene expression levels of five commonly used RGs, i.e., Beta-2-Microglobulin (B2M), ribosomal-protein-L13a (RPL13A), Peptidylprolyl-Isomerase-A (PPIA), hydroxymethylbilane synthase (HMBS), and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were quantified, and their stability was established through the RefFinder. The stability of identified RGs was validated by quantification of Perforin and granzyme B, signature molecules of renal graft dysfunction. Urine precipitated with 12% 6 K PEG yielded round and double-membraned UEVs of size ranging from 30 to 100 nm, as confirmed through transmission electron microscopy. Nanoparticle tracking analysis (59 ± 22 nm) and Dynamic-light-scattering (78 ± 56.5 nm) confirmed their size profile. Semi-quantitative Exocheck antibody array demonstrated the presence of EV protein markers in UEV. Using the comparative ΔCт method and RefFinder analysis, B2M (1.6) and RPL13A (1.8) genes emerged as the most stable reference genes. Validation of target gene expression in renal graft dysfunction patients confirmed the efficiency of B2M and RPL13A through significant upregulation compared to other RGs. Our study identified and validated B2M and RPL13A as optimal RGs for mRNA quantification studies in the UEVs of patients with renal graft dysfunction. [Display omitted] •Urinary Extracellular vesicles (UEVs) are ideal sources of liquid biopsies.•PEG-precipitation from urine is a cost-effective approach to UEV isolation.•Gene expression analysis requires reference genes (RG) for normalisation.•B2M and RPL13A are ideal RGs identified from the UEVs RNA pool.•The above RGs were validated in renal graft dysfunction patients.