Mirvana microrna isolation kit

The procedure of study material preparation and sequencing was conducted as previously described in [28 (link)].
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood specimens using density gradient centrifugation with Gradisol L reagent (Aqua-Med, Łódź, Poland). Proportions of white blood cells subpopulations in AAA group were obtained from venous blood morphology analysis results and were presented in Figure S1.
Small RNA fractions (for miRNA expression analysis) were isolated from PBMCs specimens of twenty eight AAA patients and nineteen control subjects using MirVana microRNA Isolation Kit (Ambion, Austin, TX, USA).
Total RNA specimens (for transcriptome analysis) were isolated from PBMCs samples of seven randomly selected AAA patients and seven randomly selected controls using TRI Reagent Solution (Applied Biosystems, Foster City, CA, USA).
Small RNA and transcriptome libraries were prepared using Ion Total RNA-Seq Kit v2, Magnetic Bead Cleanup Module kit, Ion Xpress RNA-Seq Barcode 01-16 Kit and sequenced on Ion 540 chips (all Life Technologies, Carlsbad, CA, USA) using Ion S5 XL System (Thermo Fisher Scientific, Waltham, MA, USA). Raw sequences of small RNA and transcriptomic libraries were aligned to 2792 human miRNAs from miRBase v21 (http://www.mirbase.org) and to 55,765 genes of hg19 human genome, respectively.

Briefly, total RNA enriched for small RNAs was isolated from whole peripheral venous blood using an mirVana microRNA Isolation Kit (Ambion, Austin, TX, USA). Reverse transcription was performed with a total reaction volume of 10 µL using miRNA-specific stem loop primers and a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Branchburg, NJ, USA) [16 (link),17 (link),18 (link),19 (link)]. Real-time qPCR reactions were performed with a total reaction volume of 15 µL consisting of 3 µL of cDNA, specific primers, TaqMan MGB probes, and the TaqMan Universal PCR Master Mix (Applied Biosystems, Branchburg, NJ, USA) in a 7500 Real-Time PCR System under standard TaqMan PCR conditions [16 (link),17 (link),18 (link),19 (link)].
MicroRNA gene expression was assessed using the delta-delta Ct method as previously described [16 (link),17 (link),18 (link),19 (link),20 (link)]. MicroRNA gene expression data were normalized to the geometric mean of the Ct values of RNU58A and RNU38B, previously selected and tested endogenous controls [21 (link)].

To determine the level of bantam in Sf9 cells, total small RNAs (<200 nt) were harvested from Sf9 cells and reverse-transcribed using miRVana microRNA Isolation Kit (Ambion, Life Technologies, Carlsbad, CA, Unites States). RT-qPCR detection of bantam was performed using miScript II RT Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. The forward primer of bantam and the internal control (U6 snRNA) were 5′-ctttctgagatcattgtgaaag-3′ and 5′-agagacgattagcatggccc-3′, respectively. The reaction was carried out on Stratagene MX3000p and data processing was done with the software MxPro. Relative expression of miRNA was normalized to U6 RNA by 2^-ΔΔt method. Similar procedures were used to determine bantam expression in S. exigua insects, except that the larvae were first ground in liquid nitrogen.

Isolation of PBMCs from whole blood samples was conducted by density gradient centrifugation using Gradisol L reagent (Aqua-Med, Poland) (see Supplementary Material).
Isolation of small RNA fractions from all PBMCs samples was performed using MirVana microRNA Isolation Kit (Ambion, Lithuania) according to the manufacturer’s protocol. The assessment of quantity and quality of isolated small RNA samples was performed using Agilent 2100 Bioanalyzer (Agilent Small RNA Kit, Agilent Technologies, Lithuania). Software implemented to Agilent 2100 Bioanalyzer was Agilent 2100 Expert Software version B.02.08.SI648.
Total RNA was isolated from PBMCs using TRI Reagent Solution (Applied Biosystems, USA), according to manufacturer’s protocol. The quantity and quality assessment of isolated total RNA was performed using Agilent 2100 Bioanalyzer (Agilent RNA 6000 Pico Kit, Agilent Technologies, USA). The RNA samples with RNA Integrity Number higher than 7 were approved for further experiments.

The intestine samples of WG and NG crayfish were delivered to the Beijing Genomics Institute-Shenzhen (BGI, Shenzhen, China) for total small RNA extraction. Briefly, total small RNA from WG and NG was extracted using the mirVana^™ micro-RNA Isolation Kit (Ambion, USA) according to the manufacturer’s protocol. The quality of small RNA samples treated with DNase I (Invitrogen, USA) was determined on a Nanodrop spectrophotometer (Nanodrop Technologies, USA). The RNA integrity number (RIN) was determined on an Agilent BioAnalyzer (Agilent Technologies, USA). RNAs with an RIN > 8.0 were chosen for small RNA library preparation and Illumina sequencing [7 (link)].

Nine milliliters of peripheral blood were collected into EDTA tubes and centrifuged twice at 1200 g for 10 min at room temperature. Plasma samples were stored at −80°C until subsequent processing.
Total RNA was extracted from 1 mL of plasma and 25 mg of normal placental tissue preserved in RNAlater (Ambion, Austin, USA), followed by an enrichment procedure for small RNAs using a mirVana microRNA Isolation kit (Ambion, Austin, USA). Trizol LS reagent was used in plasma samples for total RNA extraction from biological fluids (Invitrogen, Carlsbad, USA) and preceded the small RNAs enrichment procedure. To minimize DNA contamination, we treated the eluted RNA with 5 μL of DNase I (Fermentas International, Ontario, Canada) for 30 min at 37°C.

Total RNA was isolated using mirVana microRNA isolation kit (Ambion). miRNA assays were conducted using TaqMan miRNA assay kits or TaqMan rodent microRNA A + B cards set v3.0 (Applied Biosystems). Hierarchical clustering was performed using Cluster 3.0 [34 (link)].