AbstractObjective: Relevancy of the reference genes have not been investigated in crayfish tissue samples. Various genes have been proposed for establishing a reference level to analyze qPCR results obtained in different tissue samples. However, majority of those genes have been investigated solely in mammalian species. There is no report related to the relevancy and the efficiency of those common reference genes in crayfish tissue samples yet. The primary aim of the present work is to compare the expression level of a set of reference genes in various crayfish tissue samples to establish a reliable reference gene for quantitative expression level comparisons.Methods: cDNA copies were synthesized by RT-PCR from equalised total RNA samples of nine different tissue samples. qPCR were performed on cDNA samples by using four different primer pairs specific for β-actin, Peptidylprolyl Isomerase B, Sarcoplasmic Calcium Binding Protein and 18S ribosomal RNA genes.Results: qActin2 amplified gene fragments for β-actin in all samples. Cycle time of the amplification reaction differed substantially between the investigated tissue types. A similar difference was observed between the melting curves of the products. The primer pair PPIB amplified gene fragments for Peptidylprolyl Isomerase B only in heart, muscle and intestine samples but not in the rest of the tissue samples. Further, both amplification and melting curves for heart, muscle and intestine were different. qSCP amplified some gene fragments for Sarcoplasmic Reticulum Binding Protein only in muscle, heart and ganglion samples. The 18S_1 amplified fragments for 18S. Amplification curves the samples superimposed on each other. Melting curves of the products were very similar. Variance of the obtained data was the lowest for 18S ribosomal RNA.Conclusion: In refer to present results; it is conceivable to propose that among all the genes investigated in the present study, expression level of ribosomal 18S RNA is the most homogeneously distributed one within the body parts of the crayfish. Further, it is minimally affected by extreme biological changes such as molting. Thus, 18S Ribosomal RNA would be suggested as a reference gene to be used when investigating tissue specific expression level of a gene in the crayfish.
uMELT: prediction of high-resolution melting curves and dynamic melting profiles of PCR products in a rich web application
