Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs

Abstract

MicroRNAs (miRNAs) have emerged as promising biomarkers of disease. Their potential use in clinical practice requires standardized protocols with very low miRNA concentrations, particularly in plasma samples. Here we tested the most appropriate method for miRNA quantification and validated the performance of a hybridization platform using lower amounts of starting RNA. miRNAs isolated from human plasma and from a reference sample were quantified using four platforms and profiled with hybridization arrays and RNA sequencing (RNA-seq). Our results indicate that the Infinite® 200 PRO Nanoquant and Nanodrop 2000 spectrophotometers magnified the miRNA concentration by detecting contaminants, proteins, and other forms of RNA. The Agilent 2100 Bioanalyzer PicoChip and SmallChip gave valuable information on RNA profile but were not a reliable quantification method for plasma samples. The Qubit® 2.0 Fluorometer provided the most accurate quantification of miRNA content, although RNA-seq confirmed that only ~58% of small RNAs in plasma are true miRNAs. On the other hand, reducing the starting RNA to 70% of the recommended amount for miRNA profiling with arrays yielded results comparable to those obtained with the full amount, whereas a 50% reduction did not. These findings provide important clues for miRNA determination in human plasma samples.

Production and purification of endogenously modified tRNA-derived small RNAs

“…Furthermore, purified tsRNAs could be used for the development of amplification-independent tsRNA quantification methods, which, in combination with using synthetic tsRNA sequences, might allow determining the effects of RNA modifications on hybridization-based read-outs. The values that we have calculated after northern blotting as to how many individual stress-induced tsRNAs might exist in a single cell fit well to previously obtained values (supplemental information in [20]) although such quantifications are inherently dependent on the initial RNA concentration measurements, the outcome of which can differ by magnitudes depending on the measurement method used [67,68]. In addition, the recently reported possibility of intraand intermolecular cross-hybridization between 5ʹ tsRNA-Gly GCC and 5ʹ tsRNA-Glu CUC in extracellular space [69] could be tested for modification-dependence using purified tsRNA species from extra-cellular fluids.…”

Production and Purification of Endogenously Modified tRNA-Derived Small RNAs

“…Here, we used similar methodology to purify specific tsRNAs from human cells after exposure to tRNA fragmentation-inducing stress conditions or when ectopically expressing the mammalian anticodon nuclease Angiogenin. Our results suggest that these in vivo and although all quantifications are dependent on the initial measurements of RNA concentrations, the outcome of which can differ by magnitudes depending on the quantification method used 60,61 . In addition, the recently reported possibility of intra-and intermolecular cross-hybridization between 5' tsRNA-Gly GCC and 5' tsRNA-Glu CUC in extracellular space 62 could be tested for modification-dependence of such hybrid formation using purified tsRNAs and after determining the RNA modification patterns of secreted tsRNAs.…”

Comparison of RNA isolation and library preparation methods for small RNA sequencing of canine biofluids

“…The Qubit microRNA Assay uses specific fluorescent dyes that selectively bind to small RNAs (detection range: 50‐10000 pg/µL). Garcia‐Elias et al have reported that the Qubit microRNA Assay is better than the NanoDrop spectrophotometer and the Agilent Bioanalyzer for the evaluation of low miRNA concentrations in human plasma . Indeed, the Qiagen exoRNeasy kit yielded the highest RNA concentration in the urine samples as determined by the Qubit microRNA Assay (Table ).…”

“…Surprisingly, for serum miRNA isolation methods, we were unable to generate libraries for RNA isolated from the miRCURY Biofluids kit, even though this kit had the highest RNA concentration based on the Qubit microRNA Assay. Since the Qubit microRNA Assay detects all forms of small RNAs, this discrepancy might have been caused by the presence of other small RNA types isolated with the miRCURY Biofluids kit.…”