Nanodrop 1000 spectrophotometer v3

mRNA preparation and microinjection were performed as previously described12 (link)18 (link). In short, wild type and mutant plasmids were linearized with NheI and transcribed in vitro with T7 polymerase mMESSAGE mMACHINETM (Ambion). Xenopus laevis oocytes were treated with collagenase and defoliculated before injection18 (link). Oocytes were injected with 20 ng mutant plasmid mRNA and incubated in modified Barth’s medium (MBM, 88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO₃)₂, 0.41 mM CaCl₂, 0.82 mM MgSO₄, 2.4 mM NaHCO₃, 10 mM Hepes, 2.5 mM sodium pyruvate, 0.1 mg/ml penicillin and 0.05 mg/ml gentamycin sulfate, pH 7.5) for 4 to 5 days at 16–18 °C prior to functional assays. All chemicals were obtained from Sigma-Aldrich (Oakville, Ontario, Canada) unless otherwise stated. Same amount of water was injected into oocytes (control) for control experiments. NanoDrop 1000 Spectrophotometer V3.7 (Thermo Fisher Scientific, USA) was used to determine the concentration of mRNA of all isoforms.
Xenopus were housed, anesthetized and euthanized following protocols approved and authorized by University of Alberta Animal Research Ethics Committee.

DNA was extracted from cultures after 48–72 h of incubation. The Realpure Genomic DNA extraction kit (Real) was used to obtain purified genomic DNA and its concentration was quantified with a NanoDrop 1000 Spectrophotometer v.3.7 (Thermo Scientific), in both cases following the manufacturer’s instructions. The integrity of DNA was assessed by electrophoresis in a 1% agarose gel (8 V/cm), precast with Gel Red (Biotium) and analysed with QUANTITY ONE software (Bio-Rad), using Lambda-HindIII Digest (Takara) as molecular weight marker.

Midgut and RoB tissues (RoB = rest of the body: bodies without heads and midgut tissues) of treatment and control adult male bed bugs were dissected by hand (using fine dissection tools) 12 h after an immune challenge, and total RNA was extracted using TRizol reagent (Invitrogen) following the manufacturer’s recommendations. Midguts of bed bugs were washed three times in PCR before RNA extraction. RNA quantity and quality were assessed using Nanodrop 1000 spectrophotometer v. 3.7 (Thermo Fisher Scientific, USA), Qubit Fluorometer (Thermo Fisher Scientific, USA), gel electrophoresis, RNA ScreenTape assay (Agilent, USA), and RT-qPCR with RPL18 (internal control) (54 (link)) and CL-defensin3. Only RNA that passed this rigorous quality assessment was used for transcriptome assembly.

Sections of 5-10 μm thickness were cut from FFPE tissue samples. Tumour tissue was scraped from at least 5 slides per case and genomic DNA was subjected to overnight digestion with sodium dodecyl sulphate and proteinase K (Sigma-Aldrich, USA) at 37°C, followed by standard phenol-chloroform (1: 1) extraction and ethanol precipitation. The precipitant was suspended in 35 μl of 10 mM Tris-Cl, of pH equals to 8.0. The quality of the genomic DNA was checked (OD A260/A280 > 1.7) spectrophotometrically, using NanoDrop 1000 Spectrophotometer V3.7, Thermo SCIENTIFIC.

Genomic DNA was extracted from a whole-blood specimen for each participant using the Qiamp mini DNA extraction kit (Qiagen GmbH, Germany), following the instructions of the manufacturer. Extracted DNA was quantified by ultraviolet spectrophotometry on NanoDrop 1000 spectrophotometer V3.7 (Thermo Fisher Scientific, Wilmington, MA). The DNA was subsequently diluted to 5 ng/μl, and 5 μl of each of the DNA samples was run on 1% agarose gel to assess the quality. The extracted DNA was then stored for ~3 months at −20°C while awaiting further molecular analysis.

The panicle (5 g) was ground into powder in liquid nitrogen, and total RNA was isolated with extraction buffer as previously described. Total RNA was dissolved in distilled diethyl pyrocarbonate (DEPC) water and quantitated using a NanoDrop 1000 spectrophotometer V3.7 (Thermo Fisher Scientific; Wilmington, DE, USA) and then stored at −80ºC until miRNA isolation. Small RNAs were isolated using the PureLink miRNA Isolation Kit (K1570-01; Invitrogen/Thermo Fisher Scientific; Waltham, MA USA) according to the manufacturer’s instructions and quantitated by the NanoDrop system.