Evaluation of RNA isolation methods for microRNA quantification in a range of clinical biofluids

Background Extracellular microRNAs (miRNAs), released from cells into biofluids, have emerged as promising biomarkers for diagnostic and prognostic purposes. Several RNA isolation methods are available for the analysis of these cell-free miRNAs by RT-qPCR. Not all methods, however, are equally suitable for different biofluids. Here, we extracted total RNA from four very diverse biofluids: serum, urine, bile, and graft preservation fluid (perfusate). Four different protocols were used: a phenol-chloroform extraction and alcohol precipitation in combination with a precipitation carrier (QP) and three different column-based isolation methods, one with phenol-chloroform extraction (RN) and two without (NG and CU). For this range of clinical biofluid samples, we evaluated the potential of these different RNA isolation methods assessing recovery efficiency and the co-purification of RT-qPCR inhibiting compounds. Results Differences were observed between each of the RNA isolation methods in the recovery of cel-miR-39, a synthetic miRNA spiked in during the workup procedure, and for endogenous miRNAs. Co-purification of heparin, a known RT-qPCR inhibitor, was assessed using heparinase I during cDNA synthesis. RT-qPCR detection of synthetic miRNAs cel-miR-39, spiked in during RNA workup, cel-miR-54, spiked in during cDNA synthesis, and endogenous miRNAs was strongly improved in the presence of heparinase I for some, but not all, isolation methods. Other, co-isolated RT-qPCR inhibitors were not identified, except for biliverdin, which co-isolated from some bile samples with one of the methods. In addition, we observed that serum and urine contain compounds that enhance the binding of heparin to certain solid-phase columns. Conclusions For reliable measurements of miRNA-based biomarkers in biofluids, optimization of RNA isolation procedures is recommended as methods can differ in miRNA detection and in co-purification of RT-qPCR inhibitory compounds. Heparinase I treatment confirmed that heparin appeared to be the major RT-qPCR inhibiting compound, but also biliverdin, co-isolated from bile, could interfere with detection.

Evaluation of RNA isolation methods for microRNA quantification in a range of clinical biofluids.

BACKGROUND Extracellular microRNAs (miRNAs), released from cells into biofluids, have emerged as promising biomarkers for diagnostic and prognostic purposes. For the analysis of these cell-free miRNAs in various biofluids by RT-qPCR, several RNA isolation methods are available. However, not all methods are equally suitable for different biofluids. The aim of this study is to evaluate the potential of different RNA isolation methods in a range of clinical biofluid samples.METHODS Total RNA was isolated from serum, urine, bile, and graft preservation fluid (perfusate) using four different protocols: phenol-chloroform extraction in combination with a precipitation carrier, and three different column-based isolation methods. Co-purification of heparin, a known RT-qPCR inhibitor, was assessed using heparinase I during cDNA synthesis. Synthetic miRNAs, spiked-in during RNA workup (cel-miR-39) or during cDNA synthesis (cel-miR-54), and endogenous miRNAs were quantified using RT-qPCR.RESULTS Recovery of cel-miR-39 significantly differed between methods with miRNeasy columns providing the best overall recovery in the four biofluids tested, as was also observed for endogenous miRNAs. Contamination of RNA with heparin differed between sample type and isolation method, and could be counteracted using heparinase I. Other co-isolated RT-qPCR inhibitors were not identified, except for biliverdin which co-isolated from some bile samples with one of the methods.CONCLUSIONS For reliable measurements of miRNA-based biomarkers in biofluids, optimization of RNA isolation procedures is recommended as methods can differ in miRNA detection and co-purification of RT-qPCR inhibitory compounds. Heparinase I treatment confirmed that heparin appeared to be the major RT-qPCR-inhibiting compound but also biliverdin, co-isolated from bile, could interfere with detection.

Construction of mutant heparinase I with significantly increased specific activity

The cleavage of heparin by heparin lyases showed great potential as a cost-effective and innoxious method for producing heparin with low molecular weight (LMWH). One of the most studied and sought heparin lyase is heparinase I (HepI). However, the industrial use of HepI was largely hampered by its low specific activity and thermal stability. In this article we describe increasing in specific heparinase I activity by stepwise site-directed mutagenesis. Thus after two cycles of mutagenesis, we obtained mutant heparinase I Flavobacterium heparinum with significantly increased specific activity (25%).

Heparin sulfate is the attachment factor of duck Tembus virus on both BHK21 and DEF cells

Background Duck tembusu virus (DTMUV, genus Flaviviruses, family Flaviviridae) is an emerging flavivirus that can infect a wide range of cells and cell lines in vitro, though the initial step of virus invasion remains obscure. Methods In this study, drug treatments that including heparin, chondroitin sulfate, heparinase I, chondroitinase ABC and trypsin were applied to detect the influence of DTMUV absorption, subsequently, the copy number of viral genome RNA was analyzed by quantitative real-time PCR. The inhibition process of viral absorption or entry by heparin was determined by western blotting, and the cytotoxicity of drug treated cells was detected by cell counting kit-8. Results We found that the desulfation of glycosaminoglycans (GAGs) with sodium chlorate had a significant effect on the adsorption of DTMUV in both BHK21 and DEF cells. Based on this result, we incubated cells with a mixture of DTMUV and GAGs competition inhibitors or pre-treated cells with inhibitors, after incubation with the virus, the NS5 expression of DTMUV and viral titers were detected. The data suggested that heparin can significantly inhibit the absorption of DTMUV in a dose dependent manner but not at the step of viral entry in BHK21 and DEF cells. Meanwhile, heparinase I can significantly inhibit DTMUV attachment step. Conclusions Our results clearly proved that heparin sulfate plays an important role in the first step of DTMUV entry, viral attachment, in both BHK21 and DEF cells, which sheds light on the entry mechanism of DTMUV.