RNA ISOLATION FROM NORMAL AND CYSTIC KIDNEYS WITH PRECELLYS

RNA ISOLATION FROM NORMAL AND CYSTIC KIDNEYS WITH PRECELLYS Zemeng Wei, Michael Rehman, Stefan Somlo. Yale University, USA Ajouter les références (auteurs, journal, université…) CONTEXT The kidneys are a pair of bean-shaped organs positioned on each side of the body. They are vital for maintaining homeostasis by filtering the bloodstream, eliminating waste products, and generating urine. Blood flows into the kidneys via the renal arteries and exits through the renal veins. Each kidney is linked to a ureter, which carries urine to the bladder. Autosomal dominant polycystic kidney disease (ADPKD) is a widespread inherited condition, most commonly resulting from mutations in the genes that produce polycystin-1 (PC1) or polycystin-2 (PC2). The disease is marked by the development of numerous fluid-filled cysts within the kidneys, which can lead to complications including high blood pressure, kidney stones, repeated urinary tract infections, and eventually kidney failure. ADPKD affects around 12 million individuals globally. There is currently no cure, and cyst growth cannot be prevented. The Bertin Precellys Evolution Touch homogenizer enables the extraction of RNA from multiple tissue samples while minimizing batch effects. In this study, it was specifically used to obtain high-quality RNA for RNA sequencing from both wild-type and cystic mouse kidneys. This application note outlines the procedure for isolating high-quality RNA from mouse kidney tissue. MATERIALS • Precellys Evolution Touch (Cat No. P002511-PEVT0- A.0) • Hard Tissue homogenizing CK28-R (Cat No. P000916-LYSK0-A.0) • Kidney tissues from adult wildtype and pre-cystic C57BL/6J mice • Trizol (Thermo Fisher Scientific, Cat. no. 15596026) • RNeasy Mini Kit (Qiagen, Cat. no. 74104) Precellys Evolution Touch CONTACT sample-prep@bertin.group www.bertin-technologies.com  RNA ISOLATION FROM NORMAL AND CYSTIC KIDNEYS WITH PRECELLYS PROTOCOL • Adult mice kidneys of both wild type and pre-cystic types stored in -80 ?C were used. Kidneys were homogenized in 800 µl of Trizol by Precellys Evolution Touch with conditions of 6800 rpm for 30s twice with 30s of ice bath in between the two homogenization. • 200 µl of chloroform was then added to the mixture and centrifuged at 14 000 rpm at cold for 20 mins. • The upper aqueous layer was then transferred to a new microfuge tube and mixed with equal amount of 100% ethanol and subsequent steps were carried out as per the RNeasy Mini Kit (Qiagen,Cat. no. 74104). • Isolated RNA was then directly used for RNAseq in which 2 µg of RNA was used to prepare cDNA using iScript cDNA synthesis kit (Biorad, Cat. no. 1708890). RESULTS • The panel A below shows run of total RNA from tissue samples on Agilent 2200 Tapestation system. The plot shows robust 28S/18S ribosomal bands demonstrating excellent isolation of whole RNA pools from kidney cells. • The panel B below shows RNA quality number (RQN) of the tissue RNAs. Three plots are shows as representative images. RQN metrics from 1–10 (1=degraded, 10=intact) are used to assess RNA sample quality before downstream applications like RNA-Seq. With the consistent profile between samples, this figure can confirms excellent RNA sample quality. A) B) Figure 1. A) RNA plot of bulk RNA isolated from kidney after run on Agilent Tapestation P R E C-0 2 6-D U 1 8 4 B) Graph of quality of RNA and RQN numbers of few representative samples. CONTACT sample-prep@bertin.group www.bertin-technologies.com 2  RNA ISOLATION FROM NORMAL AND CYSTIC KIDNEYS WITH PRECELLYS CONCLUSION • The results shows that Precellys Evolution Touch is an excellent homogenizer that allow easy RNA preparation from mouse kidney tissue of normal and pre-cystic types in a midi-throughput fashion. • This is an important advance as limited midi-throughput methods exist. These methods reduce batch effects that affect downstream analysis after sequencing. See: Wei et al, Glis3 is a modifier of cyst progression in autosomal dominant polycystic kidney disease. bioRxiv, 2025 Join the Bertin Technologies community! 1500+ documents available online on the Application center www.bertin-technologies.com/application-center/ CONTACT sample-prep@bertin.group www.bertin-technologies.com 3