2.1 Red deer specimens and sampling sites
Male red deer (Cervus e. hispanicus) specimens came from three different areas in Spain: Lagunes deer farm and Cabañeros National Park (Dehesa del Carrizal), both in Ciudad Real province (Castilla La Mancha region), and El Pardo in the Community of Madrid (Supplementary Table S1). None of the animals showed clinical signs of disease. Data and blood samples were collected in 2019 and 2020 during antlers cutting, a legal traditional management activity usually conducted in July (when antlers have almost stopped growing but still have blood supply) to protect other animals and handlers from injury. Since a deer > 4 years old is considered mature, we divided our samples into two categories: young (n = 5; up to 4 years old) and adult (n = 10, 4 years and older).
Our access to these farms was approved by their management authorities. No deer were culled to carry out this study and blood was collected without the need of anaesthetize the animals thus in the present study we selected this research material as it is not an invasive sample collection method. Ethical issues were revised and approved by the Wildlife Research Unit (UIRCP-UCO), University of Cordoba. All our methods were performed in accordance with relevant guidelines and regulations. In addition, the study was carried out in accordance with ARRIVE guidelines.
2.2 Antler blood samples
Blood samples were collected from the base of the antler at the antler cut-off time (dehorned blood) in 10 ml EDTA-containing tubes and immediately transferred to RNAlater® solution (500 µL blood per 1.3 mL of preserver) for RNA stabilization.
Samples were kept at 4ºC for less than 3 days and then stored at -20ºC till use.
2.3 RNA extraction, quantification, quality measures and cDNA synthesis
Total RNA extraction from dehorned blood samples preserved in RNAlater® was carried out using the commercial RiboPureTM-Blood Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Briefly, samples (500 µL blood per 1.3 mL of preserver) were centrifuged (1 min, 16,000 xg) and the supernatant was carefully and thoroughly removed. Sample pellets were lysed with (800 μL) Lysis Solution and (50 μL) Sodium Acetate Solution and vigorously vortexed. Then, 500 μL of Acid-Phenol:Chloroform were added, samples were vortexed for 30 s and the mixture stored at room temperature for 5 min and centrifuged (1 min, 16,000 xg). The aqueous (upper) phase containing the RNA was transferred to a new tube, thoroughly mixed with 600 μL (~one-half volume) of 100% ethanol, applied in successive rounds of ~700 μL to a Filter Cartridge assembly and centrifuged (5-10 s, 16,000 xg). The RNA retained in the column was washed, first with 700 μL of Wash Solution 1 and then, twice with 700 μL of Wash Solution 2/3. Finally, the Filter Cartridge was transferred to a new tube and the RNA eluted with (50 μL) Elution Solution (preheated to ~75°C), denatured by heating at 55-60ºC for 10 minutes followed by rapid cooling on ice for at least 5 minutes, and kept at -80ºC till use. The concentration and purity of RNA preparations were determined spectrophotometrically. An Agilent 2100 Bioanalyzer (Agilent Scientific Instruments) was used to determine the integrity of the isolated RNA and assign an RNA Integrity Number (RIN) to each sample.
gDNA removal and cDNA synthesis were achieved by using the QuantiTect® Reverse Transcription Kit (Qiagen), following the manufacturer’s instructions. The cDNA was diluted to 25 ng/uL (qRT-PCR working solution) and stored at -80°C until use.
2.4 RT-qPCR
This study was carried out to conform to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments[6].
2.4.1 Selection of reference genes
Putative reference genes were selected based on information about their common use as references in the literature (Supplementary Table S2).
Primers were designed using red deer gene sequences deposited in the GenBank database (NCBI, https://www.ncbi.nlm.nih.gov/genbank/) with OLIGO 7 Primer Analysis Software (Molecular Biology Insights, Inc., http://www.oligo.net) as previously described[38]. In addition to being free of hairpins and duplex structures, primers were required to have a high Tm to ensure specificity[7]. The sequences and some characteristics of the primer pairs used for specific amplification of selected genes, as well as the PCR conditions used for transcript quantification are listed in Table 1. Table 1 also includes data about the primers specific for amplification of the genes ANXA2, APOD and TPM1, used in this work for validation of the selected reference genes. Before being used in qRT-PCR, primers specificity was evaluated by 1% agarose gel electrophoresis of their PCR amplicons, which were also sequenced and then compared with those in the GenBank database.
Table 1 Primer pairs used in this work.
Gene symbol
|
Accession number
|
Primer sequences (5´® 3´)
|
Junction
|
Amplicon length (bp)
|
E2 (R2)
|
Ref.
|
Reference genes
|
Exon
|
|
|
|
SDHA
|
OWK02252.1
|
F: CGCTCAAGGAGCCCTGCCCTGCAC
|
3
|
118
|
98.7
(98.3)
|
This work
|
R: GGCATGCCGTAATTCTCCAGGCGTCCCC
|
PGK1
|
OWJ99526.1
|
F: GGGGAAGCGGGTCGTCATGAGGATCAAGGC
|
3
|
117
|
97.6
(98.8)
|
This work
|
R: GGGACACCATCAGGCCGGCCCAGG
|
GAPDH
|
OWK03708.1
|
F: ACTGCTTGGCCCCCCTGGCCAAGGTC
|
6
|
147
|
99.6
(99.1)
|
This work
|
R: GGGCAGCCCCTCGGCCATCACGCCAC
|
ACTB
|
DQ233465.1
|
F: GATCTGGCACCACACCTTCTA
|
3
|
217
|
96,5
(97.0)
|
[34]
|
R: CCCAGAGTCCATGACAATACC
|
RPLP0
|
OWK14870.1
|
F: GCCTCACATCCGGGGGAACGTGGGCTT
|
5
|
155
|
101.4
(99.3)
|
This work
|
R: CCAGACCGGTGTTCTGGGCCGGCACAGTGA
|
GUSB
|
OWK11092.1
|
F: CTTCTCCGACAACCGGCGCCAGGGCT
|
1
|
122
|
100.1
(100)
|
This work
|
R: GCCCGTCCTGGCCCACGTCGTTGAAACTTG
|
B2M
|
OWK09923.1
|
F: CCTGCTGTCCCACGCTGAGTTCACCC
|
2
|
77
|
98.7
(100)
|
This work
|
R: CTGCGAAAGTAATGTGCTTCACTCGGCAGC
|
G6PD
|
XM_043895505.1
|
F: GGACCTCCCCGGGGCCCCGCGCCGAAAG
|
1
|
102
|
96.5
(97.3)
|
This work
|
R: CGCGAGTCCGCCTGCAGCTCCCCGCAGCTC
|
Bone growth related genes
|
|
|
|
|
APOD
|
XM_043874311.1
|
F: CGCCCTCGTGTACTCCTGTACCACGAT
|
5
|
219
|
98.2
(99.5)
|
This work
|
R: ATGGAGTGAATGCAGCTCCCTTTAGAGCCT
|
TPM1
|
DQ239919.1
|
F: GCCATTTCCCAAATTGACAT
|
14
|
211
|
95.3
(97.3)
|
[34]
|
R: CCACAGTGGGACCTTTTGTT
|
ANXA2
|
DQ239920.1
|
F: CTTCCGCAAGCTGATGGTCGCCCTCGC
|
7
|
167
|
97.5
(99.6)
|
This work
|
R: CGCTCCGCTCGGTCATGATGCTGATCCAC
|
Transcript quantification by real-time qRT-PCR was performed according to the recommendations of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE, https://www.gene-quantification.de/miqe-index.html)[6] as previously described[35]. The PCR protocol was adapted to the different Tm of each primer pair.
Genes Phosphoglycerate Kinase 1 (PGK1), Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), Ribosomal Protein Lateral Stalk Subunit P0 (RPLP0) and Glucuronidase Beta (GUSB) were amplified using a two-step protocol composed by a 98ºC, 4 min polymerase activation step and 40 cycles including a denaturation (95ºC, 15 s) step and a hybridization/extension (70ºC, 30 s) step.
A hybridization/extension (65ºC, 30 s) step was used for Succinate Dehydrogenase (SDH), Beta-2-microglobulin (B2M), Glucose-6-Phosphate 1-Dehydrogenase (G6PD), apolipoprotein D (APOD) and annexin 2 (ANXA2) genes. Finally, genes ß-Actin (ACTB) and a-tropomyosin (TPM1) were quantified with a classical three step protocols: denaturation (95ºC, 15 s), primer annealing (60ºC, 30 s) and target elongation (70ºC, 15 s). All samples were quantified in quadruplicate in a CFX-96 Touch Real-Time PCR Detection System (Bio-Rad), by using 50 ng of cDNA per reaction (20 mL) and the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad), following the manufacturer’s instructions. A melting curve analysis from 65°C to 95 °C was applied to all PCR reactions to ensure specificity of amplification in each PCR reaction.
Before reading the CT values, the absence of signal in the negative control (non-template sample) was tested. When this was the case, the CT values generated by qRT-PCR were transferred to Microsoft Excel and the mean of the quantification cycles was calculated as the average of the four replicates if the SD was less than 0.2; otherwise, the CT value of the outlying sample was removed from the study and the remaining data (at least n = 3) were reanalyzed.
The efficiency of amplification of each primer pair was evaluated by amplifying in quadruplicate a 10-fold dilution series (resulting in a concentration range from 20 to 2×105 pg) of a mixture of all cDNA samples[47]. The slope of the standard curve obtained by plotting the obtained CT values against the RNA amount per well was used to calculate the efficiency according to the equation E=10(−1/slope) −1.
2.4.2 Analysis of the expression stability of candidate reference genes
The cycle threshold (CT) values generated from qRT-PCR for all the samples and putative reference genes (Supplementary Table S3) were transferred to Microsoft Excel and used to analyze the expression stability of candidate reference genes by using the statistical algorithm software programs GeNorm[49], NormFinder[1], BestKeeper[39], the comparative ∆CT method[45] and the comprehensive web-based analysis tool RefFinder[52]. CT data in Microsoft Excel format were used to evaluate expression stability via the ΔCT method or they were directly imputed into the BestKeeper or RefFinder software. For GeNORM and NormFinder use, the CT values were first used for calculation of linear relative values (keeping the lowest relative quantity for each gene as one), which were then imported into software to calculate gene expression stability value (M) and to rank genes according to their M value. The cut-off M value was set at 1.0, with a lower M value indicating more stable expression.
2.4.3 Confirmation of the suitability of selected reference genes
Three genes related to antler growth, ANXA2, APOD and TPM1 were selected as target genes to validate the reliability of identified reference genes in blood samples of young (n = 5) and adults (n = 10) specimens of males red deer. ANXA2 and APOD can be linked to the robust development of the antler while TPM1 slows down the vigorous cell proliferation making conditions favorable toward differentiation[34].
Average CT values were calculated from all the biological replicates in each experimental condition, and from three technical replicates used for relative expression analyses. Relative quantification of target genes in different samples was carried out using the comparative ∆∆CT method according to the equation Fold variation = (1 + E)−ΔΔCT where E represent the efficiency of amplification of the primer pairs, and the most stable reference genes identified were used for normalization.
2.4.4 Statistical analysis
Comparison of data between the young (control) and the adult groups was carried out with Dunnett’s test using InStat v.3.05 (GraphPad software Inc., CA, USA). All results were evaluated using an unpaired Student’s t-test, and differences with p < 0.05 were regarded as statistically significant.