Lightcycler lc480 2 system

To analyze ADAMTS19 mRNA levels, total RNA was extracted using TRIzol Reagent (Invitrogen, Life Technologies) and used as template to synthesize cDNA using Superscript-II reverse transcriptase (Invitrogen, Life Technologies) with random hexamers for priming. We designed two primers that anneal in exon 20 (primer PB176) and exon 21 (primer PB177), generating a 242 bp amplicon. The amplification was quantified in real time using SYBR-Green Master Mix in a Lightcycler LC480-II System (Roche, CA). After 40 cycles, the specificity of the amplification was verified by melting curve analysis, and the amplicon size was subsequently confirmed by electrophoresis in 2 % (w/v) agarose gels. All reactions were performed in duplicate. Expression levels were calculated using the 2^−∆∆Ct method combining both GAPDH and TPT1 as normalization genes. In all reactions, efficiency was very close to 2 within the range of concentrations assayed.

The validation of gene expression data was performed by qRT-PCR for 14 genes (up-regulated, down-regulated and with no regulation according to microarray data) using a LightCycler® LC 480 II system (Roche). For each target gene a total of 24 experimental cDNA samples in duplicate (from 12 BLV+ and 12 BLV− individuals) and a triplicate of no template control (NTC, molecular grade water) were amplified. All procedures including cDNA synthesis, primers design, PCR reaction set up, cycling conditions, melting curve analysis, amplicon’s size and specificity control, standard curve preparation, PCR efficiency (E) and Error value calculation and quantification cycle (Cq) determination were performed as in [6 (link)].The primers and the target amplicons information are presented in Table 4. For qRT-PCR data normalization, a pair of RPLP0 and UCHL5 was employed, as the most stable reference genes for this experiment, based on a previous determination from a panel of 10 putative reference gene candidates [6 (link)]. The gene expression differences between BLV-infected and BLV uninfected groups were estimated with Relative Expression Software Tool REST 2009 V2.0.13 [37 (link)].