Table 1.
Description of cDNA synthesis kits.
Table 2.
Primer and probes with optimized temperature conditions for real-time PCR.
Fig 1.
Schematic presentation of the study design.
A) RNA of brain tissue from six naïve animals was extracted and pooled to create an identical dilution series. cDNA synthesis was then performed with each of the four kits. B) RNA of brain tissue from 6 naïve animals and 6 animals suffering traumatic brain injury was extracted. Each sample was reverse-transcribed, using all four kits, again ensuring the exact same starting material for each kit.
Fig 2.
Influence of kit selection on linearity and efficacy of cDNA synthesis.
The figure depicts absolute cDNA copy numbers after reverse transcription of one mRNA dilution series (pooled from mRNA of six naïve animals) with four different cDNA synthesis kits and RT-qPCR for the housekeeping genes ß2M and PPIA and the low-copy, regulated gene iNOS. All four kits show a linear correlation between the mRNA concentration and the cDNA output for ß2M and PPIA (Spearman's rank-order correlation coefficient; r2>0.99), except at a ß2M mRNA concentration of 1μg. Only kit 3 shows a strong linear correlation between iNOS cDNA and mRNA (r2>0.99; c). The mRNA/cDNA ratio varies significantly between the individual kits and the genes, although the individual kits show a certain consistency regarding the single gene dilution series (d-f). All plots show mean ± S.D.
Fig 3.
Influence of kit selection on absolute copy numbers.
This figure demonstrates absolute cDNA copy numbers from naïve (n = 5) vs. trauma (n = 5) samples for PPIA (high-copy, non-regulated gene), iNOS and IL-1β (both low-copy, regulated genes). An equal amount of each sample was reverse-transcribed, using all four kits. A significantly higher cDNA expression was present in trauma vs. naïve samples for PPIA (kit 2, p = 0.002; kit 3, p = 0.014; and kit 4, p = 0.027), iNOS and IL-1β (all kits, p<0.05). Absolute and relative results vary significantly between the kits used (a-c). In accordance with our previous findings, the mRNA/cDNA ratio varies substantially between the kits (d) (although identical starting material was used) and between genes (although reverse-transcribed by the same kit under equal experimental conditions). All bar charts show mean ± S.D.
Fig 4.
Impact of kit selection on expression data in an experimental paradigm of brain injury.
To demonstrate whether discrepancies in the efficacy of cDNA synthesis between the four kits influence the results after normalization, the regulated genes iNOS and IL-1β were normalized against the non-regulated PPIA. Although equal amounts of mRNA were used, even after normalization, results for iNOS and IL-1β vary significantly (a, b). After calculating the percentage of normalized trauma cDNA from normalized naïve cDNA for each kit, no significant difference between the kits could be shown neither for iNOS nor for IL-1β (c, d) (p = n.s.). All bar charts show mean ± S.D.