Dataset of proinflammatory cytokine and cytokine receptor gene expression in rainbow trout (Oncorhynchus mykiss) measured using a novel GeXP multiplex, RT-PCR assay

A GeXP multiplex, RT-PCR assay was developed and optimized that simultaneously measures expression of a suite of immune-relevant genes in rainbow trout (Oncorhynchus mykiss), concentrating on tumor necrosis factor and interleukin-1 ligand/receptor systems and acute phase response genes. The dataset includes expression values for drpt, il11a, il1b1, il1b2, il1b3, il1r-like-1(e3-5), il1r-like-1(e9-11), il1r1-like-a, il1r1-like-b, il1r2, saa, tnfa1, tnfa2, tnfa3, tnfrsf1a, tnfrsf1a-like-a, tnfrsf1a-like-b, tnfrsf5, and tnfrsf9. Gene expression was measured at four time-points post-challenge in both a resistant line (ARS-Fp-R) and a susceptible line (ARS-Fp-S) of rainbow trout. In addition, fish body weight, spleen index and the Flavobacterium psychrophilum load are reported. These data are an extension of information presented and discussed in “Proinflammatory cytokine and cytokine receptor gene expression kinetics following challenge with Flavobacterium psychrophilum in resistant and susceptible lines of rainbow trout (Oncorhynchus mykiss)” (Kutyrev et al., 2016) [1].


a b s t r a c t
A GeXP multiplex, RT-PCR assay was developed and optimized that simultaneously measures expression of a suite of immune-relevant genes in rainbow trout (Oncorhynchus mykiss), concentrating on tumor necrosis factor and interleukin-1 ligand/receptor systems and acute phase response genes. The dataset includes expression values for drpt, il11a, il1b1, il1b2, il1b3, il1r-like-1(e3-5), il1r-like-1 (e9-11), il1r1-like-a, il1r1-like-b, il1r2, saa, tnfa1, tnfa2, tnfa3, tnfrsf1a, tnfrsf1a-like-a, tnfrsf1a-like-b, tnfrsf5, and tnfrsf9. Gene expression was measured at four time-points post-challenge in both a resistant line (ARS-Fp-R) and a susceptible line (ARS-Fp-S) of rainbow trout. In addition, fish body weight, spleen index and the Flavobacterium psychrophilum load are reported. These data are an extension of information presented and discussed in "Proinflammatory cytokine and cytokine receptor gene expression kinetics following challenge with Flavobacterium psychrophilum in resistant and susceptible lines of rainbow trout (Oncorhynchus mykiss)" (Kutyrev et al., 2016)

Value of the data
Data are provided describing the optimization and validation of a new GeXP assay for measuring rainbow trout proinflammatory cytokines.
The data include PCR product relative migration and identification that are of use to researchers when performing this assay.
The gene expression values from the rainbow trout resistant line, ARS-Fp-R, and the susceptible line, ARS-Fp-S, can be used in future studies for meta-analyses comparing the response to other pathogens or environmental conditions.

Data
The dataset includes assay optimization and meta-data associated with measuring gene expression kinetics of bacterial cold water disease resistant and susceptible genetic lines of rainbow trout (Oncorhynchus mykiss) following challenge with Flavobacterium psychrophilum [1]. The GeXP assay primer dilutions were optimized and the predicted PCR products matched their elution peak in single reactions (Table 1). Kanamycin gene positive control RNA was included as an internal control for the RT and PCR reactions. Spleen samples were collected at 6 h, 24 h, 48 h and 144 h post-challenge, RNA extracted and reverse transcribed, and gene expression measured by GeXP [1]. Representative amplification profiles from 24 h samples are shown (Fig. 1A-D). Two unidentified bands were present at 253 bp and 163 bp. The complete gene expression dataset includes the meta-data of tank number, sample order, RNA extraction batch, GeXP run and normalized gene expression value (Supplemental Data Table 1). Correlation of gene expression values measured either GeXP or by syber green RT-PCR or for genes il11a ( Fig. 2A) and tnfrsf5 (Fig. 2B). Expression of tnfrsf5 was not regulated by infection [1] and thus has lower variation between samples and a lower correlation between assays [2].

Gene expression analysis
The Genome Lab GeXP genetic analysis system (Beckman Coulter Inc.; Pasadena, CA, USA) was used to quantify transcript expression. Primers were designed as described [1]. Amplicons were between 100 and 400 base pairs and each PCR product was separated in size by a minimum of three base pairs (Table 1). Twenty-one primer-pairs amplified genes associated with the immune response and four primer pairs amplified reference genes ( Table 1). The multiplex reverse transcription (RT) primer concentrations were optimized by dilution for spleen tissue ( Table 1). The size of each amplicon closely matched the expected length (Table 1).

Real time RT-PCR
One microgram of DNase treated RNA was used in a 40 μL reverse transcription reaction random primers (Invitrogen, Carlsbad, CA) and MMLV (Promega, Madison, WI) per manufacturer's protocol.
Four μL of cDNA was used in a 15 mL PCR reaction with 7.5 mL 2X SYBR Green Master Mix (ABI Biosystems, Foster City, CA), 833 nM forward primer and 833 nM reverse primer. Real-time PCR reactions were completed in duplicate for tnfrsf5 and il11a, with gapdh as a reference gene. Primers sequences were identical to those used in the GeXP multiplex assay. The real time PCR was performed with an ABI7900 Sequence Detection System (Applied Biosystems) using a 2-step PCR procedure with the following protocol. Temperature was held at 50°C for 2 min followed by 95°C for 10 min. Forty PCR Table 1 Gene product predicted size and elution peak size determined using optimized primer concentrations. The trout reference genes are arp, actb, gapdh and ef1α. Kan r RNA is an independent template used as a positive control for RT and PCR reactions.