Fig. 3. Assessment of qRT-PCR assay by determination of the copy number of exogenous RNA at a known concentration in reaction solutions. (A) Amplification plots generated by the increase of reporter dye fluorescence with each cycle of PCR of salmon GTH 2 cDNA. The solid boxes show standard solutions, and the open boxes show the test samples. All reactions were performed in duplicate (1 µl each, which was 1/15 of the total volume of the reverse-transcribed products). An arbitrary threshold cycle was obtained in the exponential phase of PCR. (B) Standard curve for the starting quantity of salmon GTH 2 RNA. The solid circles show standard solutions, and the open circles the test samples. (C) Copy numbers of salmon GTH 2 RNA in 1 µl samples (1/15 of the total volume of the reverse-transcribed product). The total volume for reverse transcription was 15 µl with 6x10⁴ RNA copies, and thus the detected value of 4143±71 copies (N=20 from the duplicates of 10 samples) in 1 µl of solution (i.e. 1/15 of the volume of the reverse-transcribed products) was reasonable. (D) Serial dilution of salmon GTH 2 RNA. The error at 10 copies shows that the S.E.M. is low enough (2.93, N=6 samples).