Table 3.
Genes | Pre-amplification | Measurements | Interpretation | |||||
---|---|---|---|---|---|---|---|---|
# Samples/comparisons | Mean Ct values | Difference observed vs expected | Manufacturer predicts success | Our findings: works? | Agreement? | |||
Observed | Expected | |||||||
CDKN1A | 0 × | 12 | 33.1 | Yes | Yes | Yes | ||
10 × | 12/12 | 23.2 | 23.1 | 0.09 | ||||
14 × | 12/12 | 19.3 | 19.1 | 0.24 | ||||
DDB2 | 0 × | 12 | 36.1 | No | No | |||
10 × | 12/12 | 27.9 | 26.1 | 1.8 | ||||
14 × | 12/12 | 24.5 | 22.1 | 2.33 | ||||
FDXR | 0 × | 12 | 37.4 | No | Yes | No | ||
10 × | 12/12 | 28.4 | 27.4 | 1.02 | ||||
14 × | 12/5 | 23.9 | 23.4 | 0.49 | ||||
GAPDH | 0 × | 12 | 33.5 | Yes | No | |||
10 × | 12/12 | 29.3 | 23.5 | 5.78 | ||||
14 × | 12/12 | 26.2 | 19.5 | 6.69 | ||||
In total | 12/89 |
Number of samples and number of comparisons per gene and pre-amplification pattern are depicted in italics. Shown are the measured/observed mean Ct values for unamplified samples as well as for samples after 10 × and 14 × pre-amplification. The column for expected mean Ct values shows the calculated Ct values when estimating a 100% linear and unbiased 10 × and 14 × pre-amplification (steps by 10 and 14, taking the mean Ct value without pre-amplification). The ΔCt (observed vs expected) is shown for each gene and each pre-amplification. We categorized the data by whether unbiased pre-amplification should have worked according to the manufacturer (Ct value without pre-amplification < 35, yes) or not (Ct value without pre-amplification > 35, no). The data are further presented if pre-amplification worked unbiased (yes) or not (no) according to the amplification biases through deviating expression patterns between amplified and un-amplified material.