| Popis: |
A Real Time PCR was set up for the detection and absolute quantification of a sea bass (Dicentrarchus labrax) cytokine gene panel. To quantify the cytokine expression by real time RT-PCR we used the standard curve method. With this method, quantification results in absolute copy number per l. Specific fragment were selected for 3 cytokine genes: IL-1, IL-6, IL-8 and for S18 of sea bass. For constructing a standard curve we used cDNA plasmid standards. Total RNA was extracted from a tissue that abundantly expressed the target genes, they were amplified by RT-PCR using the same primers as for real time RT-PCR. The amplied targets were purified, ligated in to the il pCR® 4-TOPO® VECTOR and transformed in to the TOP10 Chemically competent E. coli cells. Finally plasmid DNA was purified and linearized to generate a robust and suitable standard for long-term study purposes. The linearized plasmids were visualized and quantified by agarose gel electrophoresis using a 1 Kbp DNA ladder and the corresponding copy number/l was calculated. The Real Time PCR assay was performed with BRYTTM Green chemistry on the 7300 Real Time PCR System (Applied Biosystem). The BRYTTM Green dye is a DNA-binding molecule with a fluorescence spectrum similar to SYBR Green I. After the amplification cycle a dissociation stage was added to estimated the specificity of the products. To evaluate the limit of detection of the assays 10-fold dilutions of the plasmid were amplified. For each target genes was obtained between 0,3 and 2,4x109 copie/µl of plasmid. Confirmation of the specificity of the amplification reaction was shown by formation of a melting curve with a single peak for each target gene indicating the formation of specific PCR products. The assay allowed analytical enumeration of 24.5, 18, 16 and 3,1 copies/µl of plasmid respectively for IL-1, IL-6, IL-8 and S18. All the analysis returned an R2≥0,99 and an efficiency of 1.00 0.20. Intra-assay variability was calculated for each cytokine and resulted between ≤0,5 and ≤0,9 Std Dev Ct showing a high reproducibility. The method revealed to be very sensitive, reproducible and accurate technique for determining mRNA levels of 3 sea bass cytokines. |
