Nucleospin mirna plasma

MiRNAs were extracted from 200 µL of blood serum using NucleoSpin miRNA Plasma (Macherey-Nagel) according to the manufacturer’s protocol. Isolated miRNAs were not quantified since concentrations were below the detection limits of the NanoDrop 2000. Follow-up procedures were conducted with undiluted samples.

DNA from HBT-14 (U-87 MG) cells was extracted using the QIAmp DNA kit (Qiagen™) according to the manufacturer’s recommendation.
The miRNAs from HBT-14 (U-87 MG) cells were extracted using miRNAeasy (Qiagen™). The miRNAs from the 97 FFPE (formalin-fixed paraffin-embedded) surgically resected tumor specimens or the 8 non-tumoral brain tissues were extracted on 3 adjacent 15 μm cuts using the miRNAeasy-FFPE kit (Qiagen™, Hilden, Germany). For the tumoral specimens, morphological control was systematically carried out beforehand by a neuropathologist (ELZ) in order to guarantee that the percentage of tumor cells was greater than 70% and the absence of areas of necrosis or hemorrhage. When this was not the case, a macro-dissection of the samples was performed to determine these quality criteria. miRNAs were extracted from the seven plasma samples using NucleoSpin miRNA Plasma (Macherey-Nagel™, Düren, Luxembourg). For each sample, miRNA extraction was carried out according to the respective manufacturer’s instructions.
The integrity and quality of the purified DNA were assessed by 1% agarose gel electrophoresis, and the DNA/miRNA concentration was measured with the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Asnières-sur-Seine, France).

For the analysis of miRNA-146a expression, RNA was isolated from the serum of 12 patients before KTx and from 16 healthy controls with the use of Nucleospin miRNA Plasma (MACHEREY–NAGEL GmbH&Co.KG, Düren, Germany). Reverse transcription was conducted using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Life Technologies, Foster City, CA, USA), in accordance with the manufacturer’s protocol. The reaction was carried out in a SimpliAmpTM Thermal Cycler (Applied Biosystems, Life Technologies, Foster City, CA, USA) at 16 °C for 30 min, 42 °C for 30 min, and 85 °C for 5 min. The product of reverse transcription was stored at −20 °C until its further use. The expression of miR-146a-5p was analyzed using Real-Time PCR. The reaction was performed on a ViiaTM 7 Real-Time PCR System (Applied Biosystems, Life Technologies, Foster City, CA, USA) using the TaqMan microRNA Assay. Primers for human miR-146a-5p and U6 together with TaqMan Universal PCR Master Mix II, no UNG (Applied Biosystems, Life Technologies, Foster City, CA, USA) were used. miR-146a-5p expression was normalized to U6, which served as an endogenous, small, nuclear RNA control (TaqMan microRNA Assays, Applied Biosystems). All reactions were carried out in duplicate. Expression was calculated using the ^ΔΔCt method.

PBMCs were isolated from 6 ml of peripheral blood in ethylenediaminetetraacetic acid dipotassium salt dihydrate (K₂-EDTA) anticoagulant tubes by density gradient centrifugation at 1200×g for 10 min at room temperature with brake using Ficoll Histopaque Plus (Cytiva Sweden AB, cat# 17144003, lot #10308253) and SepMate-15 tube (STEMCELL Technologies, Canada, cat# 85415, lot#327374). Isolation of total RNA from PBMCs was performed using TRIzol RNA Isolation Reagent (Life Technologies, US, cat# 15596018, lot#19698001) and RNA concentration and purity were evaluated by NanoDrop One (Thermo Fisher Scientific Inc., US). Platelet-poor plasma was collected in K₂-EDTA-anticoagulant tubes (BD Vacutainer, cat# 368860, lot# 2203608) and isolated as previously reported [19 (link)]. Briefly, cell- and platelet-free plasma was prepared following a 2-steps centrifugation protocol: samples were initially centrifuged at 1500g for 15ʹ at 4 °C. The supernatant was collected and centrifuged again at 14,000g for 15ʹ at 4 °C and plasma aliquots were stored at − 80 °C. RNA was isolated using NucleoSpin miRNA Plasma (cat# 740981.50, lot# 2105/001, Macherey–Nagel, Germany) according to the manufacturer’s instructions.

For analysis of the miR-146a-5p expression, RNA was isolated from sera of 13 patients (before and three months after anti-TNF-α treatment) and 16 healthy controls with the use of Nucleospin^® miRNA Plasma (MACHEREY–NAGEL GmbH&Co.KG). Reverse transcription was conducted using TaqMan^® MicroRNA Reverse Trascription Kit Cat. # 4,366,596 (Applied Biosystems, Life Technologies), in accordance with the manufacture’s protocol. The reaction was carried out in a SimpliAmpTM Thermal Cycler (Applied Biosystems, Life Technologies) at 16 °C for 30 min, 42 °C for 30 min, and 85 °C for 5 min. The product of reverse transcription was stored at –20 °C until further use.
Expression of miR-146a-5p was analyzed by Real Time PCR. The reaction was performed on a ViiaTM 7 Real Time PCR System (Applied Biosystems) using the TaqMan microRNA Assay quantitate miRNAs: hsa-miR-146a-5p Cat. # 4,427,975 primers for human miR-146a-5p and U6 together with TaqMan Universal PCR Master Mix II, no UNG Cat. # 4,440,040 (Applied Biosystems). MiR-146a-5p expression was normalized to U6, which was endogenous small nuclear RNA control (TaqMan microRNA Assays, Applied Biosystems). All reactions were carried out in duplicates. The results were analyzed using the (ΔΔCt) calculations. The data are presented as mean ± SEM.

Circulating miRNA was isolated from 300 µL of plasma using NucleoSpin miRNA Plasma (Macherey-Nagel, Germany) following the manufacturer’s protocol. Before isolation 25 fm of synthetic miR-39 from C. elegans miRNA (Institute of Biochemistry and Biophysics, Polish Academy of Sciences) was added to each sample. qScript microRNA cDNA Synthesis Kit (Quantabio, MA, USA) was used for first strand cDNA synthesis; we used 4 µL of each sample for cDNA synthesis. miRNA expression was assessed using qPCR performed with Bio-Rad CFX96 Real-Time Detection System (Bio-Rad, CA, USA). Reaction solution was as follows: 1 µL of cDNA sample, iQ SYBR Green Supermix (Bio-Rad, CA, USA), Universal Primer from qScript micrRNA Synthesis Kit, specific forward primer for analyzed miRNA. Quantification of the target miRNA expression value was expressed as 2∆Ct and NormFinder algorithm was used to find the best reference gene. Ct results were normalized to spike-in C. elegans miRNA and miR-223-5p.