Sample collection and storage intervals in common fridge
Thirty-six blood samples (six samples per animal) were collected from the jugular vein from six Holstein calves, using a vacuum system (Vacutainer®, Becton Dickinson) containing EDTA anticoagulant. After collection, the blood samples were stored in a refrigerator at 4 ° C until the moment of analysis. This study was approved by the Ethics Committee on Animal Experimentation of the Instituto de Zootecnia (Protocol Nr. 298-2020).
Extraction of total RNA from whole blood
The thirty-six blood samples were divided into six groups and were submitted to RNA extraction at the following intervals post-sampling (ps): 2 hours, 1 day, 2 days, 3 days, 7 days and 14 days. The RNA extractions were performed using the protocol described by Jiang et al. , with some modifications. Each tube containing 5 mL of whole blood was centrifuged at 2,000 x g at 4 °C for 10 min. The layer of plasma was discarded, and 0.5 mL of buffy coat layer was aspirated, transferred into a new 2 mL microtube and 1.5 mL of RBC lysis buffer was added. The tube was vigorously shaken and incubated at room temperature (RT) for 15 min prior to centrifugation at 2,000 x g for 10 min (4°C). Supernatant was discarded, and 1.0 mL RBC lysis buffer was added to the cell pellet, followed by incubation at RT for 10 min. After centrifugation at 2,000 x g for 10 min, at 4 °C, supernatant was discarded, WBC pellet lysed and homogenized with 1.0 mL Qiazol® Reagent (Qiagen, Hilden, Germany). The lysate was transferred into a 1.5 mL microtube and incubated at RT for 5 min. Then 0.2 mL of 1-Bromo-3-chloropropane was added to the lysate, followed by vigorously shaken for 15 sec and incubated at RT for 5 min. The sample was centrifuged for 15 min at 12,000 x g at 4 °C. The upper layer was transferred to 1.5 mL fresh tube, mixed with 0.5 mL 100% isopropanol alcohol, and incubated at RT for 10 min. The mixture was centrifuged for 10 min at 12,000 x g at 4 °C. The pellet was washed with 1 mL 75% ethanol and centrifuged at 7,500 x g for 5 min. at 4 °C. The pellet was air dried and dissolved in 50 μL TE-buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0). The recovered RNA was stored at –80°C.
The concentration and purity of the RNA samples were determined using a BioDrop spectrophotometer (BioDrop uLITE, Integrated Scientific Solutions Inc., Walnut Creek, CA, USA). RNA integrity was verified with Agilent RNA 6000 Nano Kit (Agilent Technologies) in an Agilent 2100 Bioanalyzer instrument (Agilent Technologies) for determination of RIN (RNA Integrity Number) and by RNA analysis in 1% gel electrophoresis.
DNase digestion and cDNA synthesis
RNA samples were treated with RQ1 RNase-Free DNase (Promega, Wisconsin, US, cat. # M6101), following the manufacturer's recommendations. The cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription kit with RNase inhibitor (ref. No. 4374967, Applied Biosystems, CA, USA), following the manufacturer's information, using OligodT primers (IDT, Coralville, IA, USA). In addition to the RNase treatments, the absence of genomic DNA amplification in the RNA samples was confirmed by qPCR assays containing treated RNA instead cDNA.
Primer design and analysis of the genes
The primers for all evaluated genes were designed using the PrimerQuest software (http://www.idtdna.com/Primerquest/Home/Index). The specificities and qualities of the primers were tested using an online tool NetPrimer (http://www.premierbiosoft.com/netprimer/), OligoAnalyzer IDT (https://www.idtdna.com/calc/analyzer) and BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGETYPE=BlastSearch&LINKLOC=blasthome) and Primer-Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Details of specific primer sets are shown in Table 1.
The expression profiles of seven genes were evaluated: beta-actin (ACTB) which encodes one of the two non-muscle cytoskeletal actins, beta-2-microglobulin (B2M) which encodes a component of MHC I molecule, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) which encodes an enzyme of glycolysis pathway, peptidylprolyl isomerase A (PPIA) encodes a protein responsible by catalyzes of the cis-trans isomerization of proline imidic peptide bonds involved in many biological processes (intracellular signaling, transcription, inflammation and apoptosis), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) which encodes a central hub protein for many signal transduction pathways, cluster of differentiation 4 (CD4) which encodes a molecule expressed in the surface of T cells, macrophages, monocytes, dendritic cells and neutrophils and interferon-gama cytokine (IFN-γ) which encodes a soluble cytokine member of type II class of interferons. The algorithms used to identify the most stable genes were geNorm , NormFinder  and BestKeeper , 2004). In addition, a final classification of most stable genes was provided by using RankAggregated tool which applied a Monte Carlo cross-entropy algorithm as previous described .
The qPCR reactions were carryout on the Rotor gene Q equipment (Qiagen, Hilden, Germany) for a final volume of 10 mL that contained: 5 mL of the QuantiNova SYBR® Green RT-PCR Kit (Qiagen) master mix, 0.3 mL (10 mM) of each primer, 2.4 mL of ultra-pure water (Sigma-Aldrich) and 2 mL of cDNA. One cycle of enzymatic activation of 95 ° C was programmed for 2 minutes followed by 35 cycles of 95 ° C for 10 seconds (denaturation) and 60 ° C for 30 seconds (annealing/extension). After amplification, a melting temperature analysis was generated by raising the incubation temperature from 60 °C to 95 °C in 0.5 °C increments with a hold step of 5 sec at each increment. All reactions were run in duplicate.
The results of RNA concentrations, 260/280 nm ratios and RNA integrity number (RIN) were analyzed using “PROC GLM” procedure and the means were compared by Tukey test (p < 0.05), using the SAS (SAS Institute, Cary, NC).
Regarding the RT-qPCR results, the analysis was performed using a mixed model described by Steibel et al. , with some modifications, to jointly analyze the expression of target and reference genes: [Ygikr = Tgi + Dik + egikr ]
Where Ygikr is the Cq obtained of the gth gene (target genes: B2M, ACTB, PPIA, GAPDH, YWHAZ and IFN-γ, or the geometric mean of the Cq of the three reference genes used: B2M, GAPDH and PPIA) from the rth well (referring to the technical replicate), in a sample obtained from animal k of treatment i (2h, 1dps, 2dps, 3dps, 7dps and 14dps). Tgi is the effect of treatment i in the expression of gene g, Dik is a random sample-specific effect (common to both genes), and egirk is a residual term. The sample-specific effect, Dik, captures differences among samples that are common to both genes, particularly those that affect total mRNA concentration, such as differential extraction or amplification efficiencies among samples. The model implemented in this study was adjusted using the "Mixed" procedure of the SAS (SAS Institute, Cary, NC). The comparisons between treatments were made through the assembly of contrasts according to Steibel et al. (2009) that allowed the hypotheses to be tested at values equivalent to fold change. Comparisons were performed between 2 hps and other intervals, aiming to evaluated if storage time led to altered gene expression.