Table 1. Relative quantitation of CD4 and CD8 mRNA transcript expression in cornea and spleen using the comparative C_T method

A: Quantitative RT-PCR was performed in triplicate using 2 μl of cDNA reverse transcribed from 500 μg of total RNA from individual cornea or spleen samples (four mice for BALB/c and C57BL/6 groups and two mice for SCID group). The mean threshold cycle (C_T) value for each target (CD4 and CD8) and endogenous reference (GAPDH), representing the PCR cycle at which the ABI 7900 HT Detection System first detects a noticeable increase in reporter florescence above baseline signal, was calculated for each group. B: The δC_T values for each target were determined by subtracting the mean GAPDH C_T value from the mean CD4 and CD8 C_T values. The standard deviation of the difference was calculated from the standard deviations of the CD4, CD8, and GAPDH values by the formula (s= the square root of s₁² + s₂²). C: In this study the SCID animal group was chosen as the arbitrary constant and all expression levels are presented as relative to the SCID group. The calculation of δδC_T involved subtraction of the δC_T of the SCID sample from the δC_T of the compared BALB/c and C57BL/6 samples. The standard deviation is equivalent to the standard deviation of the δC_T. D: The range of CD4 and CD8 fold expression relative to the SCID cornea and spleen was determined by the expression: 2-δδC_T± the standard deviation as shown in Figure 4.