M mlv rt enzyme

For reverse transcription, 500 ng of total blood RNA from each sample was added to Rnase-free water (final volume 8 μl) followed by DNase I treatment for 15 min at room temperature. Samples were then added to a mix containing 4 μl of first strand 5× buffer, 2 μl of 10× dithiothreitol, 1 μl of 10 mM dNTP mix, 300 ng/μl of hexaprimer (random primers), 20–40 U/μl of RNasin® enzyme (Promega), and 200 U/μl of MMLV-RT enzyme (Life Technology). The reverse transcription reaction program consisted of three steps: 10 min at 25 °C, 50 min at 37 °C, and 15 min at 70 °C. mRNA expression was measured using a LightCycler 480 real-time PCR system and SYBR Green I Master mix® (Roche Diagnostics) (primers are listed in Additional file 5: Table S1). Standard curves were generated for each run by serial dilution of control cDNA. Gene expression levels were expressed as ratios relative to that of reference genes (GAPDH for HMOX1 and TBP for EDNRA). Real-time PCR reactions were done in duplicate in two independent reverse transcriptions.

Total RNA from M. anisopliae cells obtained under different growth conditions was extracted by standard procedures using Trizol Reagent (Life Technologies, Grand Island, NY, USA) on powder samples grinded on mortar and pestle with liquid nitrogen. Residual DNA was submitted to DNase treatment (Thermo Scientific, MA, USA) and, then, to an RNeasy Cleanup column (Qiagen, Hilden, Germany). One µg of total RNA, quantified on a Qubit fluorometer (Life Technologies, Grand Island, NY, USA), was used for cDNA synthesis using MMLV-RT enzyme (Life Technologies, Grand Island, NY, USA). The procedures were performed according to the manufacturers’ instructions, and all RNA samples were stored at –80°C. To validate chitinase gene predictions, the transcript detection was accomplished by RT-PCR. RT-PCR primers for each chitinase gene were designed for the flanking intron sequences to observe differential band patterns when compared to genomic DNA. Considering the conservation of chitinase genes, the design of oligonucleotides for PCR involved searching specific regions for each gene (Table S2).

Total RNA extraction from M. anisopliae cells harvested under
all seven different growth conditions was performed in triplicate. Samples were
ground using a mortar and pestle in liquid nitrogen, prior to standard RNA
extraction using Trizol Reagent (Life Technologies, Grand Island, NY, USA).
Residual DNA was removed with DNase (Thermo Scientific, MA, USA). Thereafter,
extracted RNAs were passed through RNeasy Cleanup columns (Qiagen, Hilden,
Germany). RNA samples were quantified using a Qubit fluorometer (Life
Technologies, Grand Island, NY, USA), and stored at -80 °C. One microgram of
total RNA was used for cDNA synthesis using MMLV-RT enzyme (Life Technologies,
Grand Island, NY, USA). All procedures were performed according to the
manufacturer’s instructions.

Total RNAs were extracted by Trizol Reagent and retro-transcribed using M-MLV RT enzyme (Invitrogen). Gene expression was quantified by real-time qPCR using Sybr Green chemistry and specific primer sets (Table A in S1 File). Classical qPCR was performed with Light Cycler 480 II using 2X SYBR Green Master Mix (Roche). High throughput qPCR was performed with BioMark HD System (Fluidigm) using Master Mix 2X EvaGreen (Bio-Rad) following a step of pre-amplification as described by the manufacturer (Fluidigm). Fold-changes were calculated using the 2^-ΔΔCT method using GAPDH gene expression levels as normalizer (S1 Fig) and one control sample as a normalizer among the different plates [4 (link),5 (link),24 (link),25 (link)]. Each sample was retro-transcribed twice independently and each RT products were quantified in triplicate at least in two independent runs of qPCR. Some genes were not amplified in some samples of series 1 and series 2 due to technical problems and the available number of samples varies from gene to gene.

According to the manufacturer’s protocol, total RNA was extracted using TRIzol LS Reagent (Thermo Fisher Scientific, USA), and concentration was measured by spectrophotometry (NanoDrop 2000 Spectrophotometer, Thermo Fisher Scientific, USA). RNA integrity was verified by electrophoresis with a 1.5% agarose gel in Tris-Borate-ethylenediaminetetraacetic acid (EDTA) buffer using GreenSafe for visualization. Total RNA from Healthy Human Astrocytes (HA) was obtained from ScienCell Research Laboratories (USA). Total RNA (1µg) was subjected to reverse transcription using the MMLV RT enzyme (Invitrogen, USA) and oligo-dT12-18 primers. Complementary DNA (cDNA) was amplified by RT-qPCR using the FastStart DNA Master SYBR Green I reagent kit for LightCycler 1.5 (Roche Diagnostics, Germany) following the manufacturer’s protocol. Primers used were the following: EZH2 (25 (link)), (FW-5’-CCCTGACCTCTGTCTTACTTGTGGA-3’, RV-ACGTCAGATGGTGCCAGCAATA-3’; 18S (FW-5’AGTGAAACTGCAATGGCTC-3’, RV-5’-CTGACCGGGTTGGTTTTGAT-3’). Relative expression of the EZH2 gene was calculated considering the 18S mRNA gene as an endogenous reference. Relative expression levels were calculated by the 2^ΔΔCt method (26 (link), 27 (link)).

Expression levels of the ABCB1 gene were evaluated prior to treatment initiation in both treatment groups. All samples were collected from the bone marrow, and the mononuclear cells were separated using Ficoll-Hypaque Hypaque (Lymphoprep, Nycomed Pharma AS, density 1.077 g/L). The mononuclear cell phase was separated and suspended in PBS medium and stored at − 70 °C. RNA Isolation was performed using TRIzol^® (Invitrogen/Life Technologies). The RNA was stored at − 80 °C until needed.
For cDNA synthesis, 2 μg of RNA, final volume of 20 μL was combined with 200 U of the MMLV RT enzyme (Invitrogen, Carlsbad, CA, USA).