RNA integrity in post-mortem samples: influencing parameters and implications on RT-qPCR assays

Abstract Messenger RNA (mRNA) profiling in postmortem human tissue might reveal information about gene expression at the time point of death or close to it. When working with post-mortem human tissue, one is confronted with a natural RNA degradation caused by several parameters which are not yet fully understood. The aims of the present study were to analyse the influence of impaired RNA integrity on the reliability of quantitative gene expression data and to identify ante-and post-mortem parameters that might lead to reduced RNA integrities in post-mortem human brain, cardiac muscle and skeletal muscle tissues. Furthermore, this study determined the impact of several parameters like type of tissue, age at death, gender and body mass index (BMI), as well as duration of agony, cause of death and post-mortem interval on the RNA integrity. The influence of RNA integrity on the reliability of quantitative gene expression data was analysed by generating degradation profiles for three gene transcripts. Based on the deduced cycle of quantification data, this study shows that reverse transcription quantitative polymerase chain reaction (RT-qPCR) performance is affected by impaired RNA integrity. Depending on the transcript and tissue type, a shift in cycle threshold values of up to two cycles was observed. Determining RNA integrity number of 136 post-mortem samples revealed significantly different RNA qualities among the three tissue types with brain revealing significantly lower integrities compared to skeletal and cardiac muscle. The body mass index was found to influence RNA integrity in skeletal muscle tissue (M. iliopsoas). Samples originating from deceased with a BMI>25 were of significantly lower integrity compared to samples from normal weight donors. Correct data normalisation was found to partly diminish the effects caused by impaired RNA quality. Nevertheless, it can be concluded that in post-mortem tissue with low RNA integrity numbers, the detection of large differences in gene expression activities might still be possible, whereas small expression differences are prone to misinterpretation due to degradation. Thus, when working with post-mortem Electronic supplementary material The online version of this articl

Apparent versus true gene expression changes of three hypoxia-related genes in autopsy derived tissue and the importance of normalisation.

The aim of our work was to show how a chosen normal-isation strategy can affect the outcome of quantitative gene expression studies. As an example, we analysed the expression of three genes known to be upregulated under hypoxic conditions: HIF1A, VEGF and SLC2A1 (GLUT1). Raw RT-qPCR data were normalised using two different strategies: a straightforward normalisation against a single reference gene, GAPDH, using the 2(-ΔΔCt) algorithm and a more complex normalisation against a normalisation factor calculated from the quantitative raw data from four previously validated reference genes. We found that the two different normalisation strategies revealed contradicting results: normalising against a validated set of reference genes revealed an upregulation of the three genes of interest in three post-mortem tissue samples (cardiac muscle, skeletal muscle and brain) under hypoxic conditions. Interestingly, we found a statistically significant difference in the relative transcript abundance of VEGF in cardiac muscle between donors who died of asphyxia versus donors who died from cardiac death. Normalisation against GAPDH alone revealed no upregulation but, in some instances, a downregulation of the genes of interest. To further analyse this discrepancy, the stability of all reference genes used were reassessed and the very low expression stability of GAPDH was found to originate from the co-regulation of this gene under hypoxic conditions. We concluded that GAPDH is not a suitable reference gene for the quantitative analysis of gene expression in hypoxia and that validation of reference genes is a crucial step for generating biologically meaningful data

Proof of concept study of age-dependent DNA methylation markers across different tissues by massive parallel sequencing

The use of DNA methylation (DNAm) for chronological age determination has been widely investigated within the last few years for its application within the field of forensic genetics. The majority of forensic studies are based on blood, saliva, and buccal cell samples, respectively. Although these types of samples represent an extensive amount of traces found at a crime scene or are readily available from individuals, samples from other tissues can be relevant for forensic investigations. Age determination could be important for cases involving unidentifiable bodies and based on remaining soft tissue e.g. brain and muscle, or completely depend on hard tissue such as bone. However, due to the cell type specificity of DNAm, it is not evident whether cell type specific age-dependent CpG positions are also applicable for age determination in other cell types. Within this pilot study, we investigated whether 13 previously selected age-dependent loci based on whole blood analysis including amongst others ELOVL2, TRIM59, F5, and KLF14 also have predictive value in other forensically relevant tissues. Samples of brain, bone, muscle, buccal swabs, and whole blood of 29 deceased individuals (age range 0-87 years) were analyzed for these 13 age-dependent markers using massive parallel sequencing. Seven of these loci did show age-dependency in all five tissues. The change of DNAm during lifetime was different in the set of tissues analyzed, and sometimes other CpG sites within the loci showed a higher age-dependency. This pilot study shows the potential of existing blood DNAm markers for age-determination to analyze other tissues than blood. We identified seven known blood-based DNAm markers for use in muscle, brain, bone, buccal swabs, and blood. Nevertheless, a different reference set for each tissue is needed to adapt for tissue-specific changes of the DNAm over time

Single lymphocytes from two healthy individuals with mitochondrial point heteroplasmy are mainly homoplasmic.

The nature of mitochondrial DNA heteroplasmy is still unclear. It could either be caused by two mitochondrial DNA (mtDNA) haplotypes coexisting within a single cell or by an admixture of homoplasmic cells, each of which contains only one type of mtDNA molecule. To address this question, single lymphocytes were separated by flow cytometry assisted cell sorting and analyzed by cycle sequencing or minisequencing. To attain the required PCR sensitivity, the reactions were carried out on the surface of chemically structured glass slides in a reaction volume of 1-2 microl. In this study, blood samples from two healthy donors showing mitochondrial point heteroplasmy in direct sequencing (195Y and 234R, respectively) were analyzed. Nearly 96% of single lymphocytes tested were found to be in a homoplasmic state, but heteroplasmic cells were also detected. These results suggest that mitochondrial point heteroplasmy in blood may well be mainly due to the mixture of homoplasmic cells

Low “penetrance” of phylogenetic knowledge in mitochondrial disease studies

An up-to-date view of the worldwide mitochondrial DNA (mtDNA) phylogeny together with an evaluation of the conservation of each site is a reliable tool for detecting errors in mtDNA studies and assessing the functional importance of alleged pathogenic mutations. However, most of the published studies on mitochondrial diseases make very little use of the phylogenetic knowledge that is currently available. This drawback has two inadvertent consequences: first, there is no sufficient a posteriori quality assessment of complete mtDNA sequencing efforts; and second, no feedback is provided for the general mtDNA database when apparently new mtDNA lineages are discovered. We demonstrate, by way of example, these issues by reanalysing three mtDNA sequencing attempts, two from Europe and another one from East Asia. To further validate our phylogenetic deductions, we completely sequenced two mtDNAs from healthy subjects that nearly match the mtDNAs of two patients, whose sequences gave problematic results

Natural radioactivity and human mitochondrial DNA mutations

Radioactivity is known to induce tumors, chromosome lesions, and minisatellite length mutations, but its effects on the DNA sequence have not previously been studied. A coastal peninsula in Kerala (India) contains the world's highest level of natural radioactivity in a densely populated area, offering an opportunity to characterize radiation-associated DNA mutations. We sampled 248 pedigrees (988 individuals) in the high-radiation peninsula and in nearby low-radiation islands as a control population. We sequenced their mtDNA, and found that the pedigrees living in the high-radiation area have significantly (P < 0.01) increased germ-line point mutations between mothers and their offspring. In each mutation case, we confirmed maternity by autosomal profiling. Strikingly, the radioactive conditions accelerate mutations at nucleotide positions that have been evolutionary hot spots for at least 60,000 years