Normalization with Corresponding Naïve Tissue Minimizes Bias Caused by Commercial Reverse Transcription Kits on Quantitative Real-Time PCR Results

<div><p>Real-time reverse transcription polymerase chain reaction (PCR) is the gold standard for expression analysis. Designed to improve reproducibility and sensitivity, commercial kits are commonly used for the critical step of cDNA synthesis. The present study was designed to determine the impact of these kits. mRNA from mouse brains were pooled to create serial dilutions ranging from 0.0625 μg to 2 μg, which were transcribed into cDNA using four different commercial reverse-transcription kits. Next, we transcribed mRNA from brain tissue after acute brain injury and naïve mice into cDNA for qPCR. Depending on tested genes, some kits failed to show linear results in dilution series and revealed strong variations in cDNA yield. Absolute expression data in naïve and trauma settings varied substantially between these kits. Normalization with a housekeeping gene failed to reduce kit-dependent variations, whereas normalization eliminated differences when naïve samples from the same region were used. The study shows strong evidence that choice of commercial cDNA synthesis kit has a major impact on PCR results and, consequently, on comparability between studies. Additionally, it provides a solution to overcome this limitation by normalization with data from naïve samples. This simple step helps to compare mRNA expression data between different studies and groups.</p></div

Influence of kit selection on linearity and efficacy of cDNA synthesis.

<p>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.</p

Impact of kit selection on expression data in an experimental paradigm of brain injury.

<p>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.</p

Schematic presentation of the study design.

<p><b>A)</b> 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>B)</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.</p

Influence of kit selection on absolute copy numbers.

<p>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.</p

Description of cDNA synthesis kits.

<p>Description of cDNA synthesis kits.</p

Expression of inflammatory marker genes and microglia activation.

<p><b>A–D:</b> The mRNA expression was determined in contused brain tissue. Expression of the inflammatory marker genes COX-2 (<b>A</b>), iNOS (<b>B</b>), and IL6 (<b>C</b>) was not significantly different between the anesthetic protocols at 15 minutes and 24 hours after experimental brain trauma (group size: naïve, n = 9; 15 minutes, n = 6; 24 hours, n = 8; data are presented as mean ± S.D.). <b>D:</b> As a marker for activated microglia, immunohistochemical analysis of Iba-1-positive cells was performed in the pericontusional tissue that revealed a significantly increased activation in sevo animals vs. comb (n = 8 per group; sevo = sevoflurane; iso = isoflurane; comb = i.p. injection of midazolam, fentanyl and medetomidine; native = no surgery; NS = not significant; p-values were adjusted for multiple comparison by Bonferroni). <b>E:</b> Example pictures of the Iba-1-stained slides of animals 24 hours post-injury (20× magnification). The number of activated microglia was determined at the border zone adjacent to the damaged brain tissue. Iba-1-positive cells are labeled dark brown (arrows).</p

Histological brain damage and neurological function.

<p>(<b>A</b>) The contusion volume was determined 15 minutes and 24 hours after experimental brain trauma (CCI). The primary lesion 15 minutes after CCI was not significantly different between the anesthetic regimens. Within 24 hours, brain contusions increased significantly in all groups. Sevo-anesthetized animals experienced a significantly larger contusion volume compared to the isoflurane and comb groups (group size: 15 minutes, n = 6 per group; 24 hours, n = 8). (<b>B</b>) The contusion areas of all groups obtained from 11 coronal brain sections (750-µm interval) cut from rostral (no. 1) to dorsal (no. 11) 15 minutes and 24 hours after CCI. The graph illustrates that the secondary expansion of brain damage occurred in all brain areas (mean ± S.E.M.). (<b>C</b>) The body weight was determined 24 hours after CCI and was compared to the value obtained before trauma as the general marker for the well-being of the animals. Animals anesthetized with comb lost significantly more body weight compared to iso. The other anesthetic regimens were not significantly different. (<b>D</b>) Neurological function was determined by the neurological severity score (NSS; 0 point = no impairment; 10 points = maximal impairment, (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019948#pone-0019948-t002" target="_blank"><b>Table 2</b></a>) 23 hours after CCI. NNS scores of comb were significantly worse compared to iso. The other groups were not significantly different. (sevo = sevoflurane; iso = isoflurane; comb = i.p. injection of midazolam, fentanyl and medetomidine; data are presented as mean ± S.D.; NS = not significant; p-values are adjusted for multiple comparison by Bonferroni).</p

Criteria of the Neurological Severity Score (NSS).

<p>One point is awarded for failure to perform a task.</p

Specific primer and probes and optimized temperature conditions for real-time PCR.

<p>Forw = sense primer; Rev = anti-sense primer, R610 = Lightcycler® Red610, Phos = phosphate, FL = fluorescein.</p