DDRT-PCR approaches applied for preeminent results in the isolation of DETs from fish brain tissues

Differential Display (DD) is a technique widely used in studies of differential expression. Most of these analyses, especially those involving fish species, are restricted to species from North America and Europe or to commercial species, as salmonids. Studies related to South American fish species are underexplored. Thus, the present work aimed to describe DD technique modifications in order to improve outcomes related to the isolation of DETs (Differentially Expressed Transcripts), using Leporinus macrocephalus, a large commercially exploited South American species, as a fish design. Different DDRT-PCR approaches were applied to brain samples and the products of the reactions were analyzed on 6% polyacrylamide gels stained with 0.17% Silver Nitrate (AgNO3). The use of PCR reactions under high stringency conditions and longer oligonucleotides based on VNTR (Variable Number of Tandem Repeats) core sequences led to better results when compared to low stringency PCR conditions and the use of decamer oligonucleotides. The improved approach led to the isolation of differentially expressed transcripts on adult males and females of L. macrocephalus. This study indicates that some modifications on the DDRT-PCR method can ensure isolation of DETs from different fish tissues and the development of robust data related to this approach.


Introduction
In the last years, diverse genetic studies have focused on the analysis of differentially expressed genes (DETs -Differentially Expressed Transcripts) or, in other words, genes expressed as messenger RNAs (mRNA) which differ in abundance among cell types or specific tissues, and which may be regulated by chemical, physiological, and environmental mechanisms (Liang and Pardee, 1995).These studies allow gene characterization and evaluation of transcript expression levels in relation to extrinsic factors.Therefore, it is possible to compare samples isolated at different developing stages, collected from different environments, or from different tissues or genders (Cervigni et al., 2008;Elis et al., 2008;Kavar et al., 2008;Alves-Costa and Wasko, 2010;Woo and Yum, 2011;Kumar et al., 2013;Dhorne-Pollet et al., 2013).
A large number of methods have been used to isolate and characterize DETs, and DD (Differential Display) (Liang and Pardee, 1992) represents one of the most popular techniques concerning this approach.The DD methodology has been widely used in differential expression studies since it shows high efficiency and is characterized for its simplicity, reduced cost and for not requiring any previous knowledge about the target genome (Liang and Pardee, 1992).The large applicability of this technique may be proven by more than 16,500 published articles, describing the use of this method (Medline 2013, January -PubMed, NCBI Web site http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed).Moreover, the differential display can simultaneously visualize increased or decreased expression of numerous mRNAs from many samples, and requires relatively small amounts of starting material (Pardee and McClelland, 1999).
Although DD also has some disadvantages, as the possibility of isolation "false-positive" transcripts (PCR products that appear to be differentially expressed on the gel but that cannot be verified when subsequent expression analyses are performed) (McClelland et al., 1995;Liang, 1998;Pardee and McClelland, 1999), technical improvements can increase its successful application.For that reason, modifications like the use of tailored primers to amplify members of a particular gene family (Jurecic et al., 1996), optimization of annealing temperature (Malhotra et al., 1998), and primer design, including the use of singleprimer correction and an annealing control primer (ACP) system (Graf et al., 1997;Kim et al., 2008, Kim et al., 2010;Ma et al., 2011), have been suggested.
Despite polymerase chain reaction (PCR)-based mRNA differential display has been widely used for identifying differentially expressed transcripts in a variety of species (e.g.Liang, 2002), some animals, as fishes, generally show a few data related to this approach.The majority of differential gene expression studies in fish species, through the use of DD, are restricted to North American and European species, or to commercial fishes, as Salmoniformes, which claim to generate important results to fish farming (Parrington and Coward, 2002).In this way, studies related to South American species are poorly exploited.Besides, works describing conditions to improve the results of fish DD are still required in this animal group.Dakis and Kouretas (2002), for example, have described some modifications in the DD technique applied to fish samples, which were characterized using a combination of longer arbitrary primers (25 and 26-mer) together with 30-mer anchored primer for PCR reactions, and the use of a nondenaturing polyacrylamide gel to analyze PCR products.
Thereby, the present work describes some different modifications of the DD technique in order to improve the results of this method in relation to DETs isolation in fish species.In an attempt to achieve this goal, we applied a DDRT-PCR approach for mRNA screening in brain tissues of Leporinus macrocephalus (piauçu), a large commercially exploited South American fish species.The improved approach permitted the isolation of differentially expressed transcripts in adult males and females of this species.

Animal samples and RNA isolation
Adult specimens of Leporinus macrocephalus (Characiformes, Anostomidae) were obtained from a private fishery station in São Paulo State, Brazil (Kabeya Fishery Station, municipality of Glicério), where they were maintained at 25 °C, and with constant aeration, until collection of brain tissues.As there is no genomic data for L. macrocephalus, the particular choice of this fish species was mainly due to the principle that the DD methodology does not require a previous knowledge on the mRNA sequences of the target biological samples.Brain tissue samples (including medulla, cerebellum, optic lobes, pineal gland, pituitary gland, cerebral hemispheres, and olfactory lobes) of 5 males and 5 females were collected and immediately stored at -80 °C until RNA extraction.
Approximately 100 mg of the brain samples were mechanically homogenized with 1 mL of TRizol Reagent (Invitrogen™) and total RNA extraction followed the manufacturer's protocol.RNA samples were eluted in RNase-free water and quantified (NanoDrop 1000 Spectrophotometer) by measuring the optical density (OD) at 260 nm.RNA purity was ensured by obtaining a 260/280 nm OD ratio ≥1.80, and its integrity was ensured by agarose gels electrophoresis.

DDRT-PCR approaches
Total RNA samples obtained from each male and female were incubated with DNase I to remove DNA contamination, which represents a further source of falsepositive bands that can be observed on differential display gels.After DNase treatment, small aliquots of each sample were loaded onto an agarose gel to check for RNA integrity.Subsequently, 2 μg high-quality RNA samples were used to prepare RNA pools of males and females, separately, in order to avoid the detection of interindividual variations not related to differences between sexes.The RNA pools were reverse transcribed (RT-PCR), separately, with the commercial kit SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen™) using an oligonucleotide (dT) 12-18 (Invitrogen™) as an anchor primer, according to the manufacturer's instructions.

Results and Discussion
Preliminary analyses were performed to determine primers that provide superior and reproducible amplification results.As so, cDNA amplification was achieved using decamer oligonucleotides commonly used in RAPD analyses and also oligonucleotides that were designed based in VNTR core sequences that correspond to minisatellite short and highly conserved regions (Jeffreys et al., 1985).The use of the 10-mer primers, that commonly generate multiple bands for different organisms (Lee et al., 2001;Yang et al., 2004;Zhang et al., 2008), did not produce this type of amplification pattern for brain tissues of L. macrocephalus.Moreover, the amplification reactions usually resulted on several faint and diffuse bands, and the results were generally not reproducible (Figure 1).These faint bands often lead to a misinterpretation of the data.In addition, just a reduced number of candidate differentially expressed transcripts could be identified when comparing males and females of L. macrocephalus through the use of RAPD primers (Figure 1).
The use of longer primers on differential display strategies may also lead to detection of DNA polymorphism through RAPD-like results.Some studies evidenced that primers with 13 bases or longer have better effects in the DD efficiency (Zhao et al., 1995;Liang, 1998;Motlik et al., 1998;Huang et al., 2001;Alves-Costa and Wasko, 2010;Alves-Costa et al., 2012).As the oligonucleotides of VNTR core sequences (14-24 bp), used as single primers to amplify the cDNA samples of L. macrocephalus, are longer than RAPD primers, the methodology could be effectively carried out at a relatively high stringency, thus yielding enhanced results -the generated bands were more distinct and the results of cDNA amplification were consistently repeatable (Figure 2).These data are consistent with the proposal that DDRT-PCR reactions need special adjustments for each species samples since its fingerprinting patterns may vary from one cell type to another (Blaes et al., 2007).
Despite this optimization in the DD approach in brain tissues of a fish species, our results led to the identification of a restricted number of amplification products, even using longer primers.The DD methodology generally leads to the visualization of 5 to 100 amplification products in polyacrylamide gel (McClelland et al., 1995).The unworkable achievement of a complex amplification pattern with a high number of bands can be due to the high PCR stringency conditions, through an annealing temperature of 55 o C, or to the use of RNA sample pools that can avoid the detection of individual variations.Despite the relative reduced number of amplified fragments (around 5 to 15 bands) visualized on polyacrylamide gels, some of them could be detected only in males or females, which indicate that they correspond to presumptive differentially expressed transcripts between the two samples.Thirty one cDNAs appeared to be differentially expressed on males and females of L. macrocephalus.Therefore, the improved DD approach using VNTR core sequences as primers in RT-PCR led to the identification of putative sex differentially expressed transcripts in this fish species and may be used as a strategy in future studies related to this approach in order to obtain consistent data.