Tissue-specific evaluation of suitable reference genes for RT-qPCR in the pond snail, [i]Lymnaea stagnalis[i]

Reverse transcription quantitative PCR (RT-qPCR) is a robust technique for the quantification and comparison of gene expression across multiple tissues. To obtain reliable results, one or more reference genes must be employed to normalize expression measurements among treatments or tissue samples. Candidate reference genes must be validated to ensure that they are stable prior to use in qPCR experiments. The pond snail (Lymnaea stagnalis) is a common research organism, particularly in the areas of learning and memory, and is an emerging target for qPCR experimentation. However, no systematic assessment of reference genes has been performed in this animal. Therefore, the aim of our research was to identify stable reference genes to normalize gene expression data from a variety of tissues in L. stagnalis. We evaluated a panel of seven reference genes across six different tissues in L. stagnalis with RT-qPCR. The genes included: elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (ACTB), beta-tubulin (TUBB), ubiquitin (UBI), prenylated rab acceptor protein 1 (Rapac1), and a voltage gated potassium channel (VGKC). These genes exhibited a wide range of expression levels among tissues. The stability of each of the genes was consistent when measured by any of the standard stability assessment algorithms: geNorm, NormFinder, BestKeeper and RefFinder. Our data indicate that GAPDH and EF1α are highly stable in the tissues that we examined (central nervous system, tentacles, lips, penis, foot, mantle) as well as in pooled analyses. We do not recommend VGKC for use in RT-qPCR experiments due to its relatively low expression stability. Our results were generally congruent with those obtained from similar studies in other molluscs. Given that a minimum of two reference genes are recommended for data normalization, we suggest GAPDH and EF1α are a strong option for multi-tissue analyses of RT-qPCR data in Lymnaea stagnalis.

and TUBB (Bavan et al., 2012;Korneev et al., 2013;Gust et al., 2013b;Flynn et al., 85 2014;Carter et al., 2015;Benatti et al., 2017). To establish a more rigorous foundation for future 86 research using RT-qPCR, we present an analysis of seven candidate reference genes across six 87 tissues of interest (tentacles, lips, foot, penis, mantle, and CNS)  98 Lymnaea stagnalis was bred and maintained in the animal care facility at StFX. The animals 99 were exposed to a photoperiod matched to natural daylight patterns in Antigonish, Nova Scotia.
100 The water in the animals' tanks was changed three times per week. The animals were fed fish 101 food and romaine lettuce ad libitum plus sinking protein pellets once per week.

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103 Total RNA Extraction 104 Total RNA was collected from a total of six biological replicates of L. stagnalis. To prepare for 105 extraction of total RNA, animals were anesthetized in in 0.125% 1-phenoxy-2-propanol 106 dissolved in Lymnaea saline for up to 30 minutes (Wyeth et al., 2009). The animals were 107 dissected to remove the shell and the digestive tract. Six tissues were isolated: CNS, tentacles, 108 lips, penis, foot and mantle. The tissues of each type from two animals were combined and then 109 flash frozen in liquid nitrogen, shredded with razor blades and weighed to ensure a mass between 110 10-50 mg. Dissected tissues were added to 500 µL of TRIzol reagent (ThermoFisher, Waltham, 111 MA). Tissue solutions were thoroughly homogenized with a PowerGen 125 tissue homogenizer 112 (ThermoFisher, Waltham, MA). Total RNA was extracted from the homogenized tissue via 113 addition of 0.5 volumes of a 24:1 solution of chloroform and isoamyl alcohol. 143 temperature between 57-62°C, a GC content between 40-60% and produce a product no more 144 than 200 bp long. Additionally, the GC content of each primer was kept within 10% of its 145 counterpart, and the melting temperature of each was kept within 3°C of its counterpart.
146 Two to six sets of primers were designed for each gene so that optimal primers could be selected 147 for the qPCR reactions. The primers were tested on combined cDNA samples to minimize tissue-148 specific bias. Melt curves were performed to verify that one product was amplified. If the 149 primers produced a single product, then 5 µL of the PCR reactions were run on a 1% (w/v) 150 agarose gel at 60 volts for roughly 30 minutes alongside a 50 bp DNA Ladder (New England 151 BioLabs). The agarose gels were analyzed with a Bio-Rad ChemiDoc (Bio-Rad Laboratories) to 152 visualize the size of the product ( Figure S5). Ultimately, one set of primers was selected for each 153 candidate reference gene based on the quality of the PCR product. The primers for each 154 candidate reference gene are listed in Table 1. 155 156 After the optimal primer sets were selected based on reaction specificity, primer efficiencies 157 were calculated for each primer set. Primer efficiency curves were generated from RT-qPCR 158 reactions on serial dilutions of RNA. The first reaction contained 100 ng total RNA and four 159 more subsequent 1:5 dilutions were performed to generate five Cq values from serial dilutions. 183 Primers for all candidate reference genes were evaluated to ensure that they could produce 184 consistent results and not amplify off-target products or generate primer dimers. EF1α was 185 measured with a specific molecular probe whereas other genes were measured with SYBR green 186 fluorescent dye. Each primer pair generated single peaks in melting curves after qPCR and single 187 bands at the correct size after electrophoresis in 1.5% agarose gels. Additionally, no 188 amplification was observed in controls that lacked reverse transcriptase in the RT-PCR or lacked 189 cDNA template in qPCR. Thus, primer pairs specifically amplified a single cDNA target. Based 190 on the standard curves, primer set efficiencies ranged from 90% (GAPDH and Rapac1) to 106% 191 (ACTB) with correlation coefficients (R 2 ) of >0.980 (Table 1).
240 Lst-GAPDH and Lst-TUBB were the top two most stable genes in the penis. Lst-EF1α and Lst-241 ACTB were the most highly ranked in the foot. Overall, Lst-GAPDH and Lst-EF1α were the 242 strongest and most consistent candidate reference gene across most tissues (Figure 4). It is also 243 worth noting that according to geNorm, Lst-EF1α was the only gene to meet its stability 244 threshold of M<1.50 in every tissue whereas Lst-GAPDH met the threshold in every tissue 245 except the mantle which had a stability value of 1.56. 246 247 Discussion 248 We assessed seven candidate reference genes in six tissues of interest (CNS, tentacles, lips, 249 penis, foot, mantle) as well as the pooled data from all tissues. There were consistent trends 250 among the genes and the rankings produced by the different algorithms were generally 251 congruent. Overall, we found Lst-GAPDH and Lst-EF1α to be the most stable pair of reference 252 genes to use in whole-body and cross-tissue comparisons of gene expression. These genes likely 253 performed exceptionally well with geNorm as they display similar expression patterns among 254 tissues. The tissue-specific analyses demonstrated some discrepancies between the algorithms 255 compared to the pooled analysis. Lst-GAPDH and Lst-EF1α were highly ranked in most tissues, 256 but Lst-VGKC was an unsuitable reference genes for every tissue. 257 258 In the context of previous research on RT-qPCR reference gene stability in molluscs, GAPDH 259 and EF1α appear to be the most favorable reference genes overall (Table 3). In one study of L.
260 stagnalis, EF1α was identified to be stable in the CNS under heat stress (Foster, Lukowiak & 261 Henry, 2015). EF1α has also been identified to be highly stable in several mollusc species and 262 thus is a strong candidate reference gene across the phylum (Wan et al., 2011;Cubero-Leon et 263 al., 2012;Moreira et al., 2014;García-Fernández et al., 2016;Huan, Wang & Liu, 2016). An 264 analysis of reference genes in the freshwater snail Bellamya aeruginosa showed that EF1α and 265 GAPDH were stable in the tentacles and penis but more variable in the foot (Liu et al., 2015), 266 similar to our findings (albeit with some discrepancies of rankings in the penis and foot tissues). 267 GAPDH has also been shown to be a stable reference gene in some molluscs (Huan, Wang & 268 Liu, 2016;Martínez-Escauriaza et al., 2018)

Figure 2
Comprehensive rankings assigned to each candidate reference gene.
Rankings were based on pooled Cq values from all tissues and assigned by geNorm, NormFinder, BestKeeper and RefFinder. *Indicates that both genes were equally recommended.

Figure 3
RefFinder comprehensive rankings for all candidate reference genes by tissue.
RefFinder calculates rankings as the geometric mean of the rankings assigned by geNorm, NormFinder and BestKeeper. Genes are ranked in order from the least stable to the most stable in each panel.

Figure 4
RefFinder comprehensive rankings for all candidate reference genes colour coded by performance.
Genes are colour coded based on stability with cyan representing the highest stability and magenta indicating the lowest while grey represents the mid-range. Description of all primers used to amplify candidate reference genes in Lymnaea stagnalis.
Primers labeled "For" are forward primers and primers labeled "Rev" are reverse primers, all sequences are written in the 5' to 3' direction. "Probe" represents the sequence of the hydrolysis probe used with the EF1α primer set. The amplification efficiency was determined from each reference gene primer set following RT-qPCR with five 1:5 serial dilutions of total RNA from 100 ng. Primers labeled "For" are forward primers and primers labeled "Rev" are reverse primers, all sequences are written in the 5' to 3' 3 direction. "Probe" represents the sequence of the hydrolysis probe used with the EF1α primer set. The amplification efficiency was 4 determined from each reference gene primer set following RT-qPCR with five 1:5 serial dilutions of total RNA from 100 ng.  Summary of the stability rankings of reference genes from studies conducted in molluscs.