Nuclease free water

All the reagents and solvents were of the highest commercially available quality and were used as received. Nuclease-free water, acrylamide/bis-acrylamide (19:1) 40% solution, Tris-Borate-EDTA (TBE) 10×, glycerol formamide and urea were purchased from VWR. Stains-All, ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) were purchased from Sigma Aldrich (Merck Life Science S.r.l., Milan, Italy). Fetal bovine serum (FBS) was provided by Euroclone (Milan, Italy).
Among the oligonucleotides studied, unmodified TBA was purchased from biomers.net GmbH (Ulm/Donau, Germany) as HPLC-purified oligomers. Their identity and purity were proved by MALDI-TOF mass spectrometry and high-performance liquid chromatography (HPLC) data, as provided by the commercial suppliers. All the TBA analogues investigated were synthesized and purified as described below. The purity of all the oligonucleotides was further confirmed by denaturing 20% PAGE analysis.

Cells (QMY23, cappz1) were lyophilized for 4 hours. The lyophilized samples were first milled by sterile toothpick, then were suspended in 1 ml TRIzol reagent (Invitrogen) and finally vortexed for 3 minutes at 25°C. Cell debris were removed by centrifugation (11750 x g, 10 min, 4°C) and the lysates were extracted by the addition 0.2 volume of chloroform (VWR) for 10 minutes. After centrifugation (11750 x g, 10 min, 4°C) the upper aqueous phase was transferred into a new tube and isopropanol (VWR) was added in 1:1 ratio. After 10 min incubation at 25°C, the precipitated RNA was collected by centrifugation (11750 x g, 10 min, 4°C) and the pellet was washed by cold 70% ethanol. After air-drying the pellets in sterile box, samples were dissolved in nuclease free water (VWR) for 10 min at 55°C. RNA concentration and purity were checked by NanoDrop (Thermo Scientific) and agarose gel electrophoresis.

Total RNA was extracted using the Quick-RNA Miniprep Plus kit (Zymo Research, Freiburg, Germany). cDNA was synthesized with the RevertAid first strand cDNA synthesis kit (Thermo Scientific) according to the manufacturer’s instructions and diluted in nuclease-free water to a final concentration of 5 ng/µL. qPCR was performed with 8 µL cDNA, 4.4 µL of 25 mM MgCl₂ (VWR), 2 µL of 10× reaction buffer (VWR) with 15 mM MgCl₂, 0.2 µL of 10 mM dNTPs (Thermo Scientific), 0.8 µL of 5 µM 6-carboxy-X-rhodamine (ROX, Eurofins, Ebersberg, Germany), 0.048 µL of 5 U/µL of Taq-Polymerase (VWR), 3.752 µL of nuclease free water (Thermo Scientific), and 0.8 µL of TaqMan primer/probe-mix (2.5 µM each, Eurofins) for each reaction. The reaction was performed with the Mastercycler Realplex (Eppendorf, Hamburg, Germany) using the following program: initial activation at 94 °C for 10 min followed by 40 amplification cycles of denaturation at 94 °C for 10 sec and annealing and extension at 60 °C for 1 min. The internal reference dye ROX was used to normalize the fluorescent reporter signal. The expressions of genes of interest were normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT). Relative gene expression was calculated by the ∆Ct and 2^−∆∆Ct methods [17 (link)]. Primers used for the reaction are listed in Table 1.

All the reagents and solvents were of the highest commercially available quality and were used as received. Nuclease-free water, acrylamide/bis-acrylamide (19:1) 40% solution, GelGreen Nucleic Acid Stain, 6X Orange DNA Loading Dye and Tris-Borate-EDTA (TBE) 10X were purchased from VWR. Formamide, urea, ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) were purchased from Sigma Aldrich. Fetal Bovine Serum (FBS) was provided by Euroclone.
Among the oligonucleotides here studied, unmodified NU172 and NU were purchased from biomers.net GmbH (Germany) as HPLC-purified oligomers. Their quality was checked by HPLC and MALDI-TOF MS by the commercial suppliers. The cyclic NU172 analogues were synthesized and purified as described below. The purity of all the oligonucleotides was further confirmed by denaturing 20% PAGE analysis.

In-hive and forager bees were placed in separate 50 mL falcon tubes and were thawed, macerated, and homogenized in 5 mL of nuclease-free water (VWR, Radnor, PA, USA) per 30 collected bees, using a 50 mL disposable tissue grinder (VWR, Radnor, PA, USA). The bee homogenate was then aliquoted as a 200 µL volume into three different 1.5 mL microcentrifuge tubes, which were stored in a −80 °C freezer until further analysis.