Inter-assay variations analysis as determined by Student’s <i>t</i>-test in expression of the five candidate reference genes for <i>T</i>. <i>parva</i> Muguga and 7014.

<p>Inter-assay variations analysis as determined by Student’s <i>t</i>-test in expression of the five candidate reference genes for <i>T</i>. <i>parva</i> Muguga and 7014.</p

Gene expression stability of five candidate <i>T</i>. <i>parva</i> reference genes as assessed by RefFinder.

<p>Gene expression stability of five candidate <i>T</i>. <i>parva</i> reference genes as assessed by RefFinder.</p

Student’s <i>t</i>-test results for analysis of intra-assay variations in the expression of HKGs between <i>T</i>. <i>parva</i> Muguga and 7014.

<p>The results from the first run are presented in panel A and results from the second run in panel B.</p

Specificity of real-time PCR amplification: Melting curves generated after amplification of five candidate <i>T</i>. <i>parva</i> reference genes showing a single melting peak for each product.

<p>Each experiment included two biological replicates of cDNA prepared from RNA isolated from cell cultures infected with <i>T</i>. <i>parva</i> Muguga and <i>T</i>. <i>parva</i> 7014 and a no template control (NTC).</p

The comparison of expression profiles of the three differentially expressed genes based on the log2 fold change values obtained from qPCR analysis when the data was normalized using 28S rRNA or β-actin as endogenous gene controls.

<p>The comparison of expression profiles of the three differentially expressed genes based on the log2 fold change values obtained from qPCR analysis when the data was normalized using 28S rRNA or β-actin as endogenous gene controls.</p

A standard curve generated by amplification of the VP2 gene from a 10-fold dilution series of BTV cDNA of known concentration (1000 to 0.1ng/μl), used to determine the concentration of <i>T</i>. <i>parva</i> cDNA by comparison with qPCR amplification of <i>T</i>. <i>parva</i> 28S rRNA from parasite isolates <i>T</i>. <i>parva</i> Muguga (designated with a red triangle) and <i>T</i>. <i>parva</i> 7014 (designated with a green triangle).

<p>A standard curve generated by amplification of the VP2 gene from a 10-fold dilution series of BTV cDNA of known concentration (1000 to 0.1ng/μl), used to determine the concentration of <i>T</i>. <i>parva</i> cDNA by comparison with qPCR amplification of <i>T</i>. <i>parva</i> 28S rRNA from parasite isolates <i>T</i>. <i>parva</i> Muguga (designated with a red triangle) and <i>T</i>. <i>parva</i> 7014 (designated with a green triangle).</p

Expression stability rankings of five candidate <i>T</i>. <i>parva</i> reference genes obtained from two qPCR runs.

<p>Actin = β-actin; Cytchr = cytochrome b; F6P = fructose-2.6-biphosphate aldolase; GAPDH = glyceraldehyde phosphate dehydrogenase.</p

Primer sequences for qPCR amplification of five candidate <i>T</i>. <i>parva</i> reference genes, the membrane transporter gene used for evaluation of qPCR assay precision and the four differentially expressed genes.

<p>Primer sequences for qPCR amplification of five candidate <i>T</i>. <i>parva</i> reference genes, the membrane transporter gene used for evaluation of qPCR assay precision and the four differentially expressed genes.</p