Mircury rna isolation kit for biofluids

Blood samples for miRNA relative expressions analysis were collected at the same time as blood samples for biochemical testing on same day when liver biopsy was performed. Serum was separated by centrifugation and stored at -80°C. MiRNAs were isolated from serum using miRCURY RNA isolation kit for biofluids (EXIQON). Before isolation, serum samples were spiked with control miRNA cel-miR-39-3p (QIAGEN), which served as control of quality of isolation and as reference gene. RNAcarrier—bacteriofage MS2 (Roche) was added for better yield.

RNA was extracted from 200 μl serum using the miRCURY RNA isolation kit for Biofluids (Exiqon #300112, Vedbaek, Denmark) according to the manufacturer’s instructions. One μg MS2 bacteriophage carrier RNA (Roche #10165948001, Castle Hill, NSW, Australia) was added during the lysis step. RNA was eluted in 25 μl nuclease-free water twice (final volume 50 μl) and stored at -80°C. RT was performed using 2 μl RNA template per 10 μl RT reaction using the Universal cDNA Synthesis kit (Exiqon #203301). Three independent RT reactions were performed per sample, and one PCR conducted for each. The real-time RT-PCR master mix contained 100-fold diluted cDNA, 1X ExiLENT SYBR Green master mix (Exiqon #203420) and 1X ROX reference dye (Life Technologies #12223–012). Ten μl of the master mix was transferred to each well of a Serum/Plasma Focus microRNA PCR panel (Exiqon #203843) or Pick-&-Mix microRNA PCR panel (Exiqon #203802) containing locked nucleic acid (LNA) primers using the epMotion 5070 liquid handling system (Eppendorf, North Ryde, NSW, Australia). Real-time RT-PCR was performed on a 7900HT Fast Real-Time PCR System (Life Technologies). PCR conditions were as follows: 95°C for 10 minutes; followed by 40 amplification cycles of 95°C for 10 seconds and 60°C for 1 minute, followed by melting curve analysis.

From 500μl serum, totalRNA was extracted using miRCURY™ RNA isolation kit for biofluids (cat#300113, Exiqon) according to manufactures protocol. TotalRNA (totRNA) quantity and quality (yield, 260/280 ratio and 260/230 ratio) were determined with the NanoDrop ND-1000 spectrophotometer (Thermo Scientific).
First strand synthesis was performed using megaplex™ RT primer pool (pool A and B, cat#4444745, Life Technologies) and TaqMan® MicroRNA reverse transcription kit (cat#4366596 Life Technologies). For each sample, 30 ng totRNA was added to the reaction mix. In order to increase the sensitivity, preamplification of the cDNA was implemented using megaplex™ preamp primer pool (pool A and B, cat#4444748, Life Technologies) and TaqMan® preamp master mix (cat#4391128, Life Technologies). The samples were diluted with 75μl of 0.1XTE pH 8.0 and then added with TaqMan® Universal PCR Master Mix, NoAmpErase® UNG, 2X. The reaction mix was applied to the TaqMan® Low Density Arrays (TLDA; Life Technologies, cat# 4444913) and loaded onto the 7900HT Fast thermocycler system (Life Technologies) for analysis. The protocol from supplier (PN 4399721) was followed for all the procedures. The microRNA microarray data have been deposited in the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo) with the accession number GSE70080.

RNA from bacterial vesicles was purified with the miRCURY™ RNA isolation kit for biofluids (Exiqon, Vedbaek, Denmark) according to the manufacturer’s protocol. One microliter of isolated RNA was examined for its quality, yield, and nucleotide length by capillary electrophoresis using an Agilent RNA 6000 Nanochip on an Agilent 2100 Bioanalyzer® (Agilent Technologies GmbH, Berlin, Germany).

Before biopsy, venous blood was drawn into coagulation tubes (Sarstedt, Nümbrecht, Germany) and further processed within two hours. Serum was prepared from the coagulated blood by centrifugation (2000 g for 10 min) and samples were stored in aliquots at −80 °C. Serum miRNAs were prepared from 200 µL of serum with the miRCURY RNA Isolation Kit for biofluids (Exiqon, Vedbaek, Denmark) in the discovery cohort and the miRNA plasma kit (Promega, Madison, WI, USA) using an automated Maxwell RSC device (Promega) in the validation cohort, according to the respective manufacturer’s recommendations.