Optimized microRNA purification from TRIzol-treated plasma

Abstract

BackgroundMicroRNAs (miRNAs) represent new and potentially informative diagnostic targets for disease detection and prognosis. However, little work exists documenting the effect of TRIzol, a common viral inactivation and nucleic acid extraction reagent, on miRNA purification. Here, we developed an optimized protocol for miRNA extraction from plasma samples by evaluating five different RNA extraction kits, TRIzol phase separation, purification additives, and initial plasma sample volume. This method was then used for downstream profiling of plasma miRNAs found in archived samples from one nonhuman primate (NHP) experimentally challenged with Ebola virus by the aerosol route.ResultsComparison of real-time RT-PCR results for spiked-in and endogenous miRNA sequences determined extraction efficiencies from five different RNA purification kits. These experiments showed that 50 μL plasma processed using the QIAGEN miRNeasy Mini Kit with 5 μg of glycogen as a co-precipitant yielded the highest recovery of endogenous miRNAs. Using this optimized protocol, miRNAs from archived plasma samples of one rhesus macaque challenged with aerosolized Ebola virus was profiled using a targeted real-time PCR array. A total of 519 of the 752 unique miRNAs assayed were present in the plasma samples at day 0 and day 7 (time of death) post-exposure. Statistical analyses revealed 25 sequences significantly up- or down-regulated between day 0 and day 7 post infection, validating the utility of the extraction method for plasma miRNA profiling.ConclusionsThis study contributes to the knowledgebase of circulating miRNA extraction methods and expands on the potential applications of cell-free miRNA profiling for diagnostics and pathogenesis studies. Specifically, we optimized an extraction protocol for miRNAs from TRIzol-inactivated plasma samples that can be used for highly pathogenic viruses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1299-5) contains supplementary material, which is available to authorized users.

Altered erythroid‐related miRNA levels as a possible novel biomarker for detection of autologous blood transfusion misuse in sport

“…Total RNA, including miRNAs, was isolated using TRIzol LS reagent (Invitrogen, Thermo Fischer Scientific), according to the manufacturer's instructions . This approach has been extensively used for microRNA analysis of plasma samples . The estimation of the isolated samples has been performed by NanoDrop Nucleic Acid Quantification procedure described by Brzobohatá et al (2016) .…”

Comparative studies of two methods for miRNA isolation from milk whey

“…the phenol-based technique (Chen et al, 2010) and column-based approach (Trizol followed by cleanup using the modified RNeasy, miRNeasy or mirVana kit) (Izumi et al, 2012). Despite some discrepancies between these two isolation methods in other body fluid samples (McAlexander et al, 2013;Moret et al, 2013;Duy et al, 2015), none of these studies compared and evaluated the quantity and quality of the isolated miRNAs from milk using these two methods.…”

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

“…To date, there is no gold standard for isolating miRNA from biofluids. Several studies have compared commercial miRNA isolation kits for serum, plasma, and urine using samples from human and nonhuman primates. In addition, various kit modifications and measurements (eg, the NanoDrop spectrophotometer [Thermo Fisher Scientific, Waltham, MA] and Agilent Bioanalyzer [Agilent Technologies, Santa Clara, CA]) have been used to compare methods.…”

“…To date, there is no gold standard for isolating miRNA from biofluids, in part due to the lack of a gold standard method for evaluation . Quantitative real‐time PCR (qRT‐PCR) is the most commonly used method for evaluating the performance of miRNA isolation methods.…”