Date: Saturday, May 30, 2020
Session Time: 3:15pm-4:00pm
Presentation Time: 3:30pm-4:00pm
*Purpose: Previously, we reported the feasibility of measuring the mRNA transcript levels of various genes in plasma exosomes of renal transplant patients for prediction of antibody-mediated rejection development by real-time (RT) qPCR. Here, on Femto Pulse system, we performed a Quality Control (QC) analysis to characterize and quantify total RNA extracted from plasma exosomes.
*Methods: Total exosome RNA was extracted from either 200 or 500 μL peripheral plasma collected from two renal transplant patients and one healthy normal individual. 6 RNA samples were analyzed on the Agilent Femto Pulse system with the FP-1201 Ultra Sensitivity RNA kit and ProSize data analysis software. Additionally, 8 exosome RNA samples from different renal transplant patients were analyzed on the Agilent Bioanalyzer system for comparison.
*Results: Total RNA yields of the 6 samples measured on Femto Pulse were 1.7±0.3 ng/200 μL plasma and 2.7±0.4 ng/500 μL plasma, similar to those in the 8 samples measured on Bioanalyzer (1.7±0.5 ng/200 μL plasma). Meanwhile Femto Pulse analysis exhibited significant superiority to Bioanalyzer for characterizing longer RNA that includes mRNA (500-6000 nucleotides [nt]) in ultra-low quantity. In longer RNA quantified by Femto Pulse (83±36 pg/200 μL plasma, 240±20 pg/500 μL plasma), 18S and 28S ribosomal RNA were consistently detected in all 6 RNA samples, while no ribosomal RNA was detected on Bioanalyzer. Finally, as the Femto Pulse analysis indicated, there was no significant difference in total exosome RNA yields and detection of longer RNA between freshly prepared plasma and archived one stored at -80°C for over 5 years with multiple freezing/thawing, confirming the superior protection to interior RNA by exosomes.
*Conclusions: RNA analysis on the Femto Pulse system suggested that full-length mRNA exist and are well protected in exosomes from peripheral plasma of renal transplant patients. This analysis and our previous RT-qPCR study confirmed that plasma exosome is ideal for mRNA transcript analysis. The Femto Pulse system exhibited superior capacity to quantify and characterize RNA including longer RNA (500-6000 nt) in ultra-low quantity.
To cite this abstract in AMA style:Zhang H, Wong K, Jordan SC, Warzak D, Toyoda M. Femto Pulse Analysis of Exosome RNA from Plasma of Renal Transplant Patients Confirmed the Unique Benefits of Plasma Exosome for Messenger RNA (mRNA) Transcript Analysis [abstract]. Am J Transplant. 2020; 20 (suppl 3). https://atcmeetingabstracts.com/abstract/femto-pulse-analysis-of-exosome-rna-from-plasma-of-renal-transplant-patients-confirmed-the-unique-benefits-of-plasma-exosome-for-messenger-rna-mrna-transcript-analysis/. Accessed May 5, 2021.
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