To confirm the accuracy, fidelity, and sensitivity of the method, we carried out several tests including the dilution of a pool of cDNA to levels equivalent to single cell content prior to measurement of gene expression in ten replicates.
The standard curve was produced from a serial dilution of a pool consisting of all cDNA samples.
For anti-pig IgA, 4 dilutions (1: 1 2,5001 3,000, 1 3,500, 1:3,500) were titrated against 100 μl of a positive control consisting of a 1 5 dilution of a pool of serum from 72 boars at DPI 21.
Ideally, with a given dilution of the pool, all QRT-PCR primer sets will amplify their perspective targets with similar efficiency.
Mice pre-pulsed with 4OH-tamoxifen were used for this set of experiments in order to allow for comparison of the dilution of the pool of GFP+ cardiomyocytes by endogenous progenitors (see below).
The working concentration of the cDNA samples was adjusted so that the amplicons would lie within the non-inhibitory concentration range defined by the 6-point standard curve which was prepared by serial dilution of a pool of equal concentrations of each cDNA, according to the guidelines presented by Gallup and Ackermann (2008).
Individual standard curves were generated for each gene using a serial dilution of a pooled skeletal muscle and a pooled adipose tissue cDNA sample.
Standard curves were constructed for each assay using serial dilution of cDNA pooled from all samples.
An aliquot of 75 μL of inhibitor dilution in the veronal buffer (Virion Serion, Würzburg, Germany) was mixed with 50 μL of antibody-sensitized sheep erythrocytes (Virion-Serion) and 25 μL of a dilution of human pooled serum in a V-microplate.
For relative quantification, standard curves were performed from a 5-step dilution series of pooled normal kidney cDNA for both gene specific and also GAPDH and ACTB reactions.
Using the 10-fold serial dilutions of a pool of these 96 RNA transcripts (pool 11 in Table 1), limit of quantification, linearity, precision and accuracy for each of RNA transcript will be determined, and the acceptance criteria of assay performance for each RNA transcript will be established.
