The study was performed on 9 m.o. male Wistar rats weighed 444 ± 69 g (M ± SD). Animal experiments were carried out under the Guidelines on the Treatment of Laboratory Animals effective at the Institute of Experimental Medicine (St. Petersburg, Russia), and these guidelines comply with EU Directive 2010/63/EU for animal experiments. Experimental protocol was approved by the Ethical committee of the Institute of Experimental Medicine. All procedures reported here were carried out in compliance with the “Animal Research: Reporting of In Vivo Experiments” (ARRIVE) guidelines.
Experimental animals were housed under standard conditions with 12 h light-dark cycle (lights on at 5 AM) and ad libitum access to water. Rats were given MCT oil (Jarrow Formulas, Inc., Los Angeles CA, USA) at a dose 2 ml/kg daily by oral gavage during a 6-hour fasting period or an equivolume of water without fasting (control animals) for 4 weeks. Animals were sacrificed by decapitation. Brains were quickly collected, frozen, and stored at -70°C for further analysis.
Brains were placed into freezing microtome Thermo-scientific™ Microm HM525 (Thermo Fisher Scientific, Waltham, MA, USA) for 60 min at -20°C and then cut in coronal direction. Medial prefrontal cortex, dorsal and ventral hippocampal regions were captured by microspatule, the structures determined based on the rat brain atlas 20, as described earlier 18. Samples were homogenized in an appropriate volume of ExtractRNA reagent (Evrogen JSC, Moscow, Russia) and total RNA was extracted by single-step acid guanidinium-thiocyanate-phenol-chloroform method 21 according to the manufacturer’s instructions.
To purify the isolated RNA from possible genomic DNA contamination, the samples were treated with ribonuclease-free deoxyribonuclease (RQ1 DNase; Promega Corp., Madison, WI, USA). The tubes with the previously precipitated RNA were centrifuged for 30 min at + 4°C, 12,000 g, and the ethanol supernatant was removed. The pellet was dried using a dry-block thermostat TS-100 (Biosan, Riga, Latvia) for 5 min at + 50°C. The dried pellet was resuspended in 7.5 µl of milli-Q water and incubated at + 50°C for 5 min for better dissolution of nucleic acids. A reaction mixture containing 10 U of a recombinant RNasin Ribonuclease Inhibitor (Sileks, Moscow, Russia) and 2 U of RQ1 DNase in a 2× reaction buffer was added to the resulting solution. The tubes with the reaction mixture were incubated for 10 min at + 37 °С. After incubation, 1.5 µL (1/10 of the volume of the reaction mixture) of a stop reagent (20 mM EGTA) was added to the samples, mixed, incubated at + 70°C to inactivate the enzyme for 5 min, followed by cooling to + 4°C.
To purify the resulting preparation from the reaction components, RNA was precipitated: the volume of the solution was brought to 30 µL with milli-Q water, 3 µL (1/10 of the volume of RNA solution) of 3M sodium acetate solution, and 75 µL (2.5 volumes of RNA solution) of 96% ethanol, thoroughly mixed, and then incubated at − 20 °С overnight. After that, the tubes were centrifuged, the precipitate was washed with 75% ethanol and stored under alcohol until further handling.
Before carrying out the reverse transcription (RT) reaction, the tubes with RNA were centrifuged for 30 min at + 4°C, 12,000 g, the RNA sediment was dried, and then dissolved in 15 µL of milli-Q water. RNA concentration (via 260 nm absorption) and purity (260/230 and 260/280 nm absorption ratios) were measured spectrophotometrically using a Nanodrop 2000 instrument (Thermo Fisher Scientific, Waltham, MA, USA). To carry out the RT reaction, 0.5 µg oligo(dT) and 0.25 µg random-9-mer primers (BioBeagle, St. Petersburg, Russia) were added to 8 µL of the RNA solution containing 1 µg of RNA preliminarily equalized in terms of concentration and incubated at + 70°С for 5 min. Samples were briefly cooled and centrifuged. Then a reaction mixture containing M-MLV reverse transcriptase (Promega Corp., Madison, WI, USA), a mixture of dNTPs (25 mM dATP, 25 mM dCTP, 25 mM dGTP, 25 mM dTTP; Medigen, Novosibirsk, Russia), 5× M-MLV reaction buffer (250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl2, 50 mM DL-dithiothreitol, pH 8.3; Promega Corp., Madison, WI, USA), RNasin in deionized water was added. The samples were thoroughly vortexed and incubated for 2 h at + 42°C for the synthesis of cDNA on the RNA template and for 10 min at + 65°C to stop the reaction through enzyme inactivation. The resulting cDNA solution (20 µL) was diluted 5-fold with deionized water and stored at − 20°C until real-time polymerase chain reaction (qPCR) with fluorescent probes (TaqMan technology).
In the present work, we tested a panel of 9 reference genes that regulate different cell functions and are frequently used for RT-qPCR data normalization in the rat brain 1. We used three triplex qPCR assays validated in our previous work 22: Actb + Gapdh + B2m; Rpl13a + Sdha + Ppia; Hprt1 + Pgk1 + Ywhaz. The descriptions of the used primer/probes are summarized in supplementary files (Table S1).
The Multiplex qPCR reactions had been optimized and fully described in our previous study 22. Briefly, the reaction mix contained 1 µL of cDNA sample, 0.75 U of TaqM-polymerase (Alkor Bio Group, St. Petersburg, Russia), 200 nM of specific forward and reverse primers, and either 200nM (for Actb) or 100 nM (for the rest of the genes) TaqMan probes, 3.5 mM MgCl2, 250 µM dATP/dTTP/dCTP/dGTP in 10 µL total volume of 1× TaqM-reaction buffer. All oligonucleotides were synthesized by DNA-Synthesis Ltd. (Moscow, Russia). All reactions were duplicated with no reverse transcription and no template control. The reactions were run on C1000 Touch Thermal Cycler combined with CFX96 Real-Time detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The thermal settings were as follows: 1 cycle at 95°C for 15 min (as recommended by the enzyme manufacturer), 5 cycles (without plate read) with a denaturation step (95°C for 5 s) and an annealing/elongation step (60°C for 10 s) and then 35 cycles with a denaturation step (95°C for 5 s) and an annealing/elongation step (60°C for 10 s) followed by fluorescence plate read (about 13 s).
The PCR curves were analyzed with the CFX Manager software (Bio-Rad Laboratories, Inc., Hercules, CA, USA): the quantification cycle (Cq) values were determined by setting a single threshold. We then imported the raw mean Cq data to RefFinder® online tool (https://www.heartcure.com.au/reffinder/) to evaluate the expression stability of the examined genes. RefFinder® utilizes four commonly used algorithms for reference gene expression stability analysis (the comparative Delta-Ct method, BestKeeper, NormFinder, and GeNorm) and calculates comprehensive ranking by geometric averaging of obtained ranks 23. Briefly, (a) in the analysis by the comparative Delta-Ct method, the selection of the most stable reference genes is achieved by comparing the relative expression of pairs of genes in each sample: genes with the smallest mean standard deviation of delta-Ct are considered the most stable 13; (b) the BestKeeper tool selects the most stable genes by ranking the geometric means of raw Cq values of each gene, comparing them in pairs, and calculating the coefficient of variation, the standard deviation of the Cq values (the lower, the more stable), and the correlation coefficient (the higher, the more stable) 14; (c) NormFinder's principle of operation is based on a mathematical model that calculates the stability value of candidate reference genes by estimating both the total variation of the reference candidate genes and the variation between subgroups of samples in a sample set 15; (d) the basic principle behind the GeNorm algorithm is to calculate the stability value M (the lower, the more stable) by averaging the pairwise variations among all candidate genes in all samples 16.