Stability of endogenous reference genes in postmortem human brains for normalization of quantitative real-time PCR data: comprehensive evaluation using geNorm, NormFinder, and BestKeeper

Abstract

In forensic molecular pathology, quantitative real-time polymerase chain reaction (RT-qPCR) provides a rapid and sensitive method to investigate functional changes in the death process. Accurate and reliable relative RT-qPCR requires ideal amplification efficiencies of target and reference genes. However, the amplification efficiency, changing during PCR, may be overestimated by the traditional standard curve method. No single gene meets the criteria of an ideal endogenous reference. Therefore, it is necessary to select suitable reference genes for specific requirements. The present study evaluated 32 potential reference genes in the human brain of 15 forensic autopsy cases using three different statistical algorithms, geNorm, NormFinder, and BestKeeper. On RT-qPCR data analyses using a completely objective and noise-resistant algorithm (Real-time PCR Miner), 24 genes met standard efficiency criteria. Validation of their stability and suitability as reference genes using geNorm suggested IPO8 and POLR2A as the most stable ones, and NormFinder indicated that IPO8 and POP4 had the highest expression stabilities, while BestKeeper highlighted ABL1 and ELF1 as reference genes with the least overall variation. Combining these three algorithms suggested the genes IPO8, POLR2A, and PES1 as stable endogenous references in RT-qPCR analysis of human brain samples, with YWHAZ, PPIA, HPRT1, and TBP being the least stable ones. These findings are inconsistent with those of previous studies. Moreover, the relative stability of target and reference genes remains unknown. These observations suggest that suitable reference genes should be selected on the basis of specific requirements, experiment conditions, and the characteristics of target genes in practical applications.

Role of Circadian Clock Genes in Sudden Cardiac Death: A Pilot Study

“…For the present study, samples were generally assessed within 2 days of death. Furthermore, we have not observed changed in mRNA levels when samples are saved at -80 degrees 24,36) . It is important that we consider about fundamental assumption underlying this study concerning accurate timing of the fatal event.…”

“…Thermal cycling conditions included 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 10 min, and 40 cycles of amplification at 95°C for 15 s and 60°C for 1 min. The threshold cycle (Ct) was calculated automatically by the instrument software with a threshold value of 0.2 24) .…”

“…Survival time was the period from the onset of the fatal insult until death. For this purpose, we used only cases where the insult was witnessed and/or circumstantial evidence was well established to confi rm survival and postmortem times, conforming to estimates based on the classic pathologic fi ndings 20,[22][23][24][25] . In this study, none of the patients were known to have been receiving medical care.…”

Quality Control of Expression Profiling Data

“…Attempts made to improve the quality of samples and to control for tissue-specificity can be exemplified by the investigations into the effect of RNA degradation post-mortem [62,63]. Fixing protocols may prevent mRNA decay but have a potential to introduce their own artefacts into the results [64,65].…”

“…While quality control methods were previously suggested, they only focussed on the genome (potentially missing alternatively spliced variants that would have been detected by studying the transcriptome) [80,81], or on the proteome (potentially missing mRNAs whose translation is downregulated by microRNAs [83,84] or covered aspects of the data such as GC content [40], noise [12,13] source material quality [62,63,67,70,86], different experimental methods [64][65][66][71][72][73][74][76][77][78][79] or read quality [87][88][89][90][91][92][93][94][95], with few investigations focusing on the tissuespecificity issues [75,96], even when two or more methods were used in the same study [97][98][99][100][101]. A common shortcoming of many previous attempts is that tissue specificity of the genes was reported [102][103][104][105][106][1...…”