Cfx thermocycler

Total bacterial RNA was extracted from 2 mL of bacterial culture or infected cells using the Total RNA Mini Plus (AABiotech, Gdynia, Poland) according to the manufacturer’s instructions. Residual DNA was digested for 30 min at 37 °C in a total volume of 20 µL using DNase I (AABiotech). Total RNA was quantified by A260 measurements (DS-11 FX; DeNovix), verified by agarose gel electrophoresis, and diluted to 1 µg/µL before cDNA synthesis. First-strand cDNA was synthesized using the iScript (Bio-Rad) according to the manufacturer’s instructions. The relative amounts of mRNA were quantified by qPCR using the CFX thermocycler (Bio-Rad) and EvaGreen (IMMUNIQ). The reaction mixture contained 10x polymerase buffer without magnesium (Pol Buffer A; EURx), 2.5 mM MgCl₂, 5 mM dNTPs, 20x EvaGreen Dye, 2.5 units of Optitaq DNA Polymerase (EURx), and 0.1 mM of each primer. cDNA was amplified as follows: 5 min incubation at 95 °C for initial denaturation, followed by 35 cycles of 20 s denaturation at 95 °C, 20 s annealing at 56 °C, and 15 s elongation at 72 °C. The target gene was normalized using 16S RNA as a reference. The comparative Cq method was used for the relative quantification of gene expression. All experiments were performed at least three times, and triplicate samples were analyzed in each experiment to confirm the accuracy and reproducibility of the qPCR.

Except when indicated, total RNA was prepared using the MasterPure RNA purification kit (Epicentre) according to the manufacturer’s instructions, except that 250 μg of proteinase K was used for cell lysis. For the experiment shown in Fig. 2A, TRIzol (Sigma) was used. Briefly, adherent cells were directly lysed with 0.1 mL/cm² TRIzol during 10 min, and the lysate was harvested before adding 0.1 mL of chloroform/mL of TRIzol. After centrifugation, the upper phase was collected and the Epicentre kit was used for the DNase digestion and subsequent precipitation. After total nucleic acids recovery, DNA was removed with a cocktail of DNase I and Baseline Zero DNase supplemented with Riboguard RNase inhibitor for 45 min at 37°C.
A 500-ng amount of total RNA was reverse transcribed with SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. A condition without SuperScript III was included for each sample and analyzed by qPCR using GAPDH exon 9 primers to check for DNA contamination. qPCR was performed in triplicate with TB green Premix Ex Taq from TaKaRa and the Bio-Rad CFX thermocycler. GADPH mRNA expression was used to normalize the results of RT-qPCR. Primers are listed in Table 3.

NZYol Reagent (NZYtech, Lisbon, Portugal) was used to extract total RNA from 10 whole specimens at 30 dpf. DNase I treatment (Promega, Madison, WI, USA) was performed with 1 µg RNA for 30 min at 37 °C and the RNA was reverse-transcribed at 37 °C for one hour using M-MLV reverse transcriptase (Invitrogen, Waltham, MA, USA), RNaseOUT (Invitrogen) and oligo-d(T) primer [5′-ACGCGTCGACCTCGAGATCGATG(T)13-3′]. qPCR assays were carried out using a Bio-Rad CFX thermocycler (Bio-RAD, Hercules, CA, USA). Gene expression levels were normalized using eef1a1l1 as a housekeeping gene [50 (link)], and the ∆∆Ct method was applied to determine relative quantification [38 (link),51 (link)]. The sequence of primers used in this study is listed in Table 1.

RNA used for assays included RNA used for Illumina RNAseq library preparation and additional biological replicates. For each genotype, n ≥ 3 biological replicates, each replicate consisting of four retinas from one male and one female mouse, were collected. RNA isolation, cDNA preparation, and qRT-PCR reactions were performed as previously described [21 (link)]. Briefly, retina tissue was immediately processed for RNA using the PerfectPure RNA tissue kit (5 Prime) and quantified. cDNA was synthesized from 1 μg of RNA using the Transcriptor First Strand cDNA Synthesis kit (Roche Applied Science). A 10 μl qRT-PCR reaction mixture containing 1× EvaGreen with Low Rox reaction mix (BioRad), 1 μM primer mix, and diluted cDNA was prepared and run on a BioRad CFX thermocycler in triplicate. Data were analyzed using QBase software (Biogazelle). Relative gene expression was normalized to Ubb and Tuba1b and FC from WT was determined. Kruskal-Wallis and Dunn's multiple comparison tests were used to determine significant FC differences from WT (p < 0.05). Primer sets were designed using MacVector software and synthesized by IDT DNA technologies (Additional file 19).

Total RNA from hippocampus of adult male and female mice (n = 6/group) was isolated using STAT 60 reagent (Tel-Test, Friendswood, TX, USA) and RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. RNA concentration was determined by absorbance at 260 nm using a spectrophotometer (Nanodrop 2000, ThermoFisher Scientific, Madison, WI, USA). Total RNA was reverse transcribed into cDNA using the iScript cDNA synthesis kit (BioRad, Hercules, CA, USA). Real-time qPCR was performed on the CFX thermocycler (BioRad, Hercules, CA, USA) using the SsoAdvanced Universal SYBR Green Supermix (BioRad, Hercules, CA, USA) using standard protocols [17 (link),54 (link),55 (link),56 (link)]. Biological replicate samples were run in triplicate. Quantification of candidate gene expression levels was calculated based on the threshold cycle (C_t) for each well using the provided software and normalized to PPP2r2p as endogenous control. Relative changes in gene expression were normalized to the control male group. To extend the findings from our RNA-Seq analysis, we designed primers for genes in categories overrepresented in our Gene Ontology analysis. Primer sequences are listed in Table 2.

Total RNA was isolated from epididymal white and interscapular brown adipocytes using the Qiagen lipid tissue kit (Cat no. 74804, Qiagen). cDNA was generated using 1 μg aliquots of the RNA (iScript, Thermo Scientific). The expression of specific mRNA was determined using real‐time PCR (CFX thermocycler, BioRad) using Taqman assays (Life Technologies) for TSHR (Mm01337707_m1), glyceraldehyde 3‐phosphate dehydrogenase (GAPDH Mm99999915_g1), uncoupling protein‐1 (UCP1 m00494069_m1), peroxisome proliferator‐receptor gamma (PPARG Mm00440940_m1), CCAAT/enhancer‐binding protein alpha (CEBPA Mm00514283_s1), hormone‐sensitive lipase (LIPE Mm00495359_m1), beta‐1adrenergic receptor (ADRB1 Mm00431701_s1), beta‐3 adrenergic receptor (ADRB3 Mm00442669_m1), solute carrier family 2/glucose transporter type 4 (SLC2A4 Mm00436615_m1), insulin receptor substrate 1 (IRS1 Mm01278327_m1), adiponectin (ADIPOQ Mm00456425_m1), and leptin (LEP Mm00434759_m1). All gene expression assays were normalized to GAPDH expression. Relative gene expressions were calculated using the delta‐delta Ct method (Livak & Schmittgen, 2001). For each experimental group (n = 6–8), expression levels were expressed as a fold‐change compared to WT mice fed on CD.