Substantial performance discrepancies among commercially available kits for reverse transcription quantitative polymerase chain reaction: A systematic comparative investigator-driven approach

Sieber, Matthias; Recknagel, Peter; Glaser, F.; Witte, Otto W.; Bauer, Michael; Claus, Ralf A.; Frahm, Christiane

doi:10.1016/j.ab.2010.03.007

Cited by 37 publications

(45 citation statements)

References 24 publications

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“…For qPCR, equal amounts of total RNA (1 µg) were transcribed in cDNA with RevertAid First Strand cDNA synthesis kit (Fermentas [27]). Amplification products were analyzed using gel electrophoresis, melting curve analysis, and sequencing to confirm the PCR product specificity.…”

Section: Methodsmentioning

confidence: 99%

Attenuated Inflammatory Response in Aged Mice Brains following Stroke

et al. 2011

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BackgroundIncreased age is a major risk factor for stroke incidence, post-ischemic mortality, and severe and long-term disability. Stroke outcome is considerably influenced by post-ischemic mechanisms. We hypothesized that the inflammatory response following an ischemic injury is altered in aged organisms.Methods and ResultsTo that end, we analyzed the expression pattern of pro-inflammatory cytokines (TNF, IL-1α, IL-1β, IL-6), anti-inflammatory cytokines (IL-10, TGFβ1), and chemokines (Mip-1α, MCP-1, RANTES) of adult (2 months) and aged (24 months) mice brains at different reperfusion times (6 h, 12 h, 24 h, 2 d, 7 d) following transient occlusion of the middle cerebral artery. The infarct size was assessed to monitor possible consequences of an altered inflammatory response in aged mice. Our data revealed an increased neuro-inflammation with age. Above all, we found profound age-related alterations in the reaction to stroke. The response of pro-inflammatory cytokines (TNF, and IL-1β) and the level of chemokines (Mip-1α, and MCP-1) were strongly diminished in the aged post-ischemic brain tissue. IL-6 showed the strongest age-dependent decrease in its post-ischemic expression profile. Anti-inflammatory cytokines (TGFβ1, and IL-10) revealed no significant age dependency after ischemia. Aged mice brains tend to develop smaller infarcts.ConclusionThe attenuated inflammatory response to stroke in aged animals may contribute to their smaller infarcts. The results presented here highlight the importance of using aged animals to investigate age-associated diseases like stroke, and should be considered as a major prerequisite in the development of age-adjusted therapeutic interventions.

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Section: Methodsmentioning

confidence: 99%

Attenuated Inflammatory Response in Aged Mice Brains following Stroke

et al. 2011

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“…It is important to emphasize that the variation introduced by each step is strictly dependent on the kit used, as different commercially available kits will exhibit differences in reproducibility (7, 9) and consequently, the CVs determined here are merely representative. Never theless, our findings clearly indicate that among the variables studied, biofilm growth made the greatest contribution to the variability detected in gene expression quantification.…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, it is important to understand the origin of such variability in order to be able to mitigate it. Variability and bias in the quantification of gene expression has been attributed to biological factors, including ( i ) RNA degradation (5), ( ii ) the presence of inhibitors (6), and ( iii ) the nature of the sample used (7), as well as non-biological factors such as ( i ) the use of different RNA extraction procedures (8), ( ii ) complementary DNA (cDNA) synthesis, and ( iii ) quantitative PCR (qPCR) kits (9, 10). Here, we measured variability associated with all three key steps in gene expression quantification: RNA extraction, cDNA synthesis, and qPCR.…”

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confidence: 99%

Assessing and Reducing Sources of Gene Expression Variability in Staphylococcus Epidermidis Biofilms

2014

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Gene expression quantification can be a useful tool in studying the properties of bacterial biofilms. Unfortunately, techniques such as RNA extraction, cDNA synthesis, and quantitative PCR (qPCR) can introduce variability into mRNA transcript measurements, obscuring biologically relevant results. Here we sought to identify the steps that impair accurate gene expression quantification from Staphylococcus epidermidis biofilm samples. We devised an experimental setup that could be used to determine the contribution of each experimental step to the variability of mRNA transcript measurement. Among factors tested, biofilm growth contributed the most bias to gene expression quantification. Additional experiments demonstrated that pooling biofilms together reduced this variability, resulting in more accurate gene expression analysis results. We therefore recommend pooling in order to reduce the variability associated with gene expression quantification from biofilm samples.

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“…RNA concentration and integrity were analyzed using Nanodrop spectrophotometer and QIAxcel microcapillar electrophoresis apparatus and was followed by reverse transcription of 1.0 μg RNA using standard conditions (Thermo Scientific) (36). The expression profile of selected mRNA was measured using the Rotor-Gene Q 2plex system.…”

Section: Discussionmentioning

confidence: 99%