Validation of Isolation Methodology and Endogenous Control Selection for qRT-PCR Assessment of Microrna Expression in Serum and Urine Exosomes

Introduction: MicroRNAs are short RNA molecules that control ~50% of genes by binding to the mRNA 3’ UTR and repressing translation. Recently, they have been detected within exosomes; small vesicles secreted by most cells and abundant in body fluids. Exosomes are highly enriched for specific microRNAs and have been proposed as the starting point for circulating biomarker studies.

To increase the accuracy of microRNA assessment by qRT-PCR, endogenous controls are required to correct for variability factors. Exosomal microRNA studies can be problematic, as endogenous controls previously used in cellular samples may not be present. This study compared exosome isolation and RNA extraction methods from urine and serum samples and identified suitable endogenous controls for incorporation into qRT-PCR analysis.

Methods and Results: For serum exosomes, specialist isolation reagents from System Biosciences (SBI) (ExoQuick Serum Exosome Precipitation Solution) and Life Technologies (Total Exosome Isolation Reagent) were compared, followed by RNA extraction (Norgen Biotek Total RNA Purification kit) and qRT-PCR assessment of 3 endogenous controls (HY3, RNU48 & U6). Superior exosomal RNA recovery was achieved using Life Technologies reagent, demonstrated by higher RNA concentration (Life Technologies ng/ul 4.4, 7.5 & 6.9 vs. SBI ng/ul 3.8, 5.0 & 2.7) and lower endogenous control Ct values (HY3: Life Technologies 25.56, 28.54 & 26.69 vs. SBI 27.48, 30.48 & 35.36. RNU48: Life Technologies 30.95, undetected & 34.45 vs. SBI 30.95, undetected & undetected. U6: Life Technologies 21.83, 24.72 & 22.59 vs. SBI 21.59, 27.55 & 32.71, respectively). Recovery of exosomes (30-150 nm) was verified by electron microscopy.

Serum exosomal RNA recovery was further assessed by isolating exosomes then comparing three commercially available RNA extraction kits (SBI SeraMir Exosome RNA Purification Column kit, Norgen Biotek Total RNA Purification kit & Qiagen RNeasy Micro kit). The Norgen Biotek kit gave the highest RNA yield (SBI ng/ul 13.0, 10.9 & 6.7 vs. Norgen ng/ul 23.2, 22.6 & 33.2 vs. Qiagen ng/ul 0.3, 0.6 & 0.4) and expression of two endogenous controls (HY3 & U6) (HY3: Norgen 26.76, 29.37 & 27.66 vs. SBI 31.45, 29.43 & 33.38 vs. Qiagen 35.00, 35.12 & 33.99. U6: Norgen 21.38, 24.96 & 21.31 vs. SBI 25.95, 24.91 & 30.17 vs. Qiagen 26.48, 27.14 & 27.39). In each case, exosomal isolation was confirmed by electron microscopy.

To validate the methodology to isolate urine exosomal RNA, a commercially available kit was compared to ultracentrifugation. The Urine Exosome RNA Isolation kit (Norgen Biotek) gave superior results compared to ultracentrifugation followed by RNA extraction using the Norgen Biotek Total RNA Purification kit. This was demonstrated by higher RNA quantity (Norgen ng/ul 6.6, 6.4 & 11.5 vs. ultracentrifugation ng/ul 3.3, 4.5 & 2.9) and endogenous control (HY3 & U6) expression (HY3: Norgen 25.31, 26.33 & 26.85 vs. ultracentrifugation 31.54, 29.21 & 29.36. U6: Norgen 31.66, 30.83 & 33.47 vs. ultracentrifugation 32.49, 33.46 & 33.30). Exosomes isolated by the Norgen kit were also visualised by electron microscopy for further validation.

The stability of 8 endogenous controls (RNU6B, RNU19, RNU38B, RNU43, RNU48, HY3, U6 & miR-320) was assessed by qRT-PCR in a test serum (n=10) and urine (n=15) exosome cohort from healthy controls and hematopoietic stem cell transplantation (HSCT) patients. HY3 and U6 were selected as the optimal controls for serum exosome miRNA expression analysis, with the highest level of stability across the panel (HY3: S.D 1.77 & CoV 6.2%, U6: S.D 2.14 & CoV 8.6%). HY3 and RNU48 were selected as the optimal controls for urine exosome miRNA expression analysis panel (HY3: S.D 1.67 & CoV 6.4%, RNU48: S.D 1.85 & CoV 5.3%).

Selected optimal controls were analysed in a clinical HSCT serum (n=55) and urine (n=50) cohort. Expression stability was acceptable for all controls (serum U6: S.D 2.93 & CoV 11.8%. HY3: S.D 2.22 & CoV 7.4%. Urine RNU48: S.D 2.26 & CoV 6.9%, HY3: S.D 2.42 & CoV 8.8%), indicating constitutive expression in clinical samples.

Conclusions: Exosomal microRNA studies are in their infancy and the number of commercially available exosome and RNA isolation kits are increasing. This study identifies the optimal methods to isolate serum and urine exosomal RNA as well as suitable endogenous controls for incorporation into qRT-PCR studies.