Abstract: TH-PO758
Isolation of High-Quality RNA from Human Veins and Arteriovenous Fistulas
Session Information
- Hemodialysis: Vascular Access - I
November 02, 2017 | Location: Hall H, Morial Convention Center
Abstract Time: 10:00 AM - 10:00 AM
Category: Dialysis
- 603 Hemodialysis: Vascular Access
Authors
- Selman, Guillermo, Albany Medical College, Albany, New York, United States
- Santos, Nieves, University of Miami, Miller School of Medicine, Miami, Florida, United States
- Martinez, Laisel, University of Miami, Miller School of Medicine, Miami, Florida, United States
- Duque Ballesteros, Juan Camilo, University of Miami, Miller School of Medicine, Miami, Florida, United States
- Tabbara, Marwan, University of Miami, Miller School of Medicine, Miami, Florida, United States
- Salman, Loay H., Albany Medical College, Albany, New York, United States
- Vazquez-Padron, Roberto I., University of Miami, Miller School of Medicine, Miami, Florida, United States
Background
Functional genomics and transcriptome analysis of pre-access veins and arteriovenous fistulas (AVF) require RNA of high quality and integrity. However, the vast majority of intraoperative vascular biopsies have a small size, are low in cell density, and rich in collagen and other extracellular matrix components that hinder the complete disruption of the tissue by guanidine isothiocyanate-based RNA extraction buffers. The application of current extraction protocols to AVF samples typically results in low RNA yields with poor quality. In this work, we developed a standard operating procedure that combines a pulverizing method that keeps the tissue completely frozen for RNA extraction with commercial reagents for purification.
Methods
We optimized this method using RNA-later (Qiagen) preserved veins (n=63) and AVFs (n=30) that were obtained intraoperatively during the creation of two-stage brachiobasilic fistulas in consented patients at the University of Miami. Briefly, 50-60 mg of tissue was cut in small pieces and ground to a fine powder in a Spex/Mill 6770 (15 min pre-cooling; 30 sec run; 2 min cycle cooling; 15 cycles total, 10 Hz rate) in the presence of 100 uL of Trizol. The homogenate was collected with 700 uL Trizol, transferred to 2-mL RNase-free microcentrifuge tubes, and further homogenized using an Ultra-Turrax T8 instrument for 30-45 seconds. After removing cellular debris by centrifugation (12,000 x g, 5 min, RT), the clear homogenate was extracted with chloroform according to the standard Trizol protocol, and 0.55 volumes of ethanol were added prior to loading onto an Omega EZNA column for further purification (Omega Bio-tek). On-column DNase digestion was applied as desired. Total RNA was eluted with 40 uL of RNAse-free water.
Results
The total RNA yield had a median of 44.5 ng/ul (interquartile range [IQR] 32.1 – 68.0) in veins and 124.0 ng/ul (IQR 85.2 – 220.0) in AVFs. High-quality RNA (RIN > 5) was obtained in 79.4% of veins and 86.7% of AVFs as demonstrated using an Agilent 2100 Bioanalyzer.
Conclusion
In conclusion, we have developed a new protocol for isolation of high-quality RNA from small vascular biopsies. We have successfully used these RNAs for RT-PCR and highly sensitive techniques for transcriptome analysis (RNA-seq).
Funding
- NIDDK Support