TaqMan PCR assay in the control of RNA normalization in human post-mortem brain tissue

The brain tissue obtained after death is subjected to several circumstances that can affect RNA integrity. The present study has been directed to reveal possible pitfalls and to control RNA normalization in post-mortem samples in order to recognize the limitations and minimize errors when using TaqMan PCR technology. This has been carried out in samples of the frontal cortex in a series of control and diseased cases covering Parkinson's disease, dementia with Lewy bodies pure form and common form, and Alzheimer's disease. Special attention has been paid to the value of the agonal state, post-mortem delay and pH of the nervous tissue as approximate predictors of the quality of RNA, as well as to the use of the Bioanalyzer to confirm RNA preservation. In addition, since possible disease-modified mRNAs have to be normalized with ideal unaltered RNAs, TaqMan human endogenous control plates have been used to determine the endogenous control most appropriate for the study. beta-glucuronidase (GUS) and beta-actin were good endogenous controls because their expression levels showed a small variation across a representative number of control and pathological cases. RNA stability was also analysed in a paradigm mimicking cumulative delay in tissue processing. GUS mRNA levels were not modified although beta-actin mRNA levels showed degradation at 22 h. Finally, the control of RNA degradation for the normalization of genes of interest was also tested. mRNA expression levels for superoxide dismutase 1 (SOD1) and metalloproteinase domain 22 (ADAM22) were examined at several artificial post-mortem times, and their expression levels compared with those for putative controls beta-actin and GUS. In our paradigm, the expressions of SOD1 and ADAM22 were apparently not modified when normalized with beta-actin. Yet their expression levels were reduced with post-mortem delay when values were normalized with GUS. Taken together, these observations point to practical consequences in TaqMan PCR studies. Short post-mortem delays and acceptable pH of the brain are not sufficient to rule out RNA degradation. The selection of adequate endogenous controls is pivotal in the study. beta-actin and GUS are found to be good endogenous controls in these pathologies, although GUS but not beta-actin expression levels are preserved in samples with long post-mortem delay.     

Biochemical and molecular studies using human autopsy brain tissue

The use of human brain tissue obtained at autopsy for neurochemical, pharmacological and physiological analyses is reviewed. RNA and protein samples have been found suitable for expression profiling by techniques that include RT-PCR, cDNA microarrays, western blotting, immunohistochemistry and proteomics. The rapid development of molecular biological techniques has increased the impetus for this work to be applied to studies of brain disease. It has been shown that most nucleic acids and proteins are reasonably stable post-mortem. However, their abundance and integrity can exhibit marked intra- and intercase variability, making comparisons between case-groups difficult. Variability can reveal important functional and biochemical information. The correct interpretation of neurochemical data must take into account such factors as age, gender, ethnicity, medicative history, immediate ante-mortem status, agonal state and post-mortem and post-autopsy intervals. Here we consider issues associated with the sampling of DNA, RNA and proteins using human autopsy brain tissue in relation to various ante- and post-mortem factors. We conclude that valid and practical measures of a variety of parameters may be made in human brain tissue, provided that specific factors are controlled.     

Pre-and Postmortem Influences on Brain RNA

Abstract: Many potentially valuable techniques for the understanding of human neurobiological and neuropathological processes require the use of RNA obtained from postmortem tissue. As with earlier neurochemical studies, there are two particular problems posed by such tissue in comparison with tissue from experimental animals. These are the postmortem interval and the condition of the patient prior to death, referred to as the agonal state. We review the nature and extent of the effects of postmortem interval and agonal state on RNA in brain tissue, with particular reference to the study of neuropsychiatric disorders. Perhaps surprisingly, postmortem interval has at most a modest effect on RNA. Abundant intact and biologically active RNA is present in tissue frozen 36 h or more after death. Postmortem interval does not account for the marked variability observed among human brains in all RNA parameters. Despite the overall stability of RNA after death, some evidence suggests that individual RNAs may undergo postmortem decay. Less attention has been paid to the effects of agonal state. The existing data indicate that events in the premortem period such as hypoxia and coma can affect the amount of some messenger RNAs. The nature of agonal state influences depends on the messenger RNA in question, though the basis for this selective vulnerability is unknown. No agonal state effect on overall RNA level or activity has been found. The data show that postmortem brain tissue can be used for RNA research. However, considerable attention must be paid to controlling for the influences of pre-and postmortem factors, especially when quantitative analyses are performed.     

DIFFERENTIAL EFFECTS OF AGONAL STATUS ON MEASUREMENTS OF GABA AND GLUTAMATE DECARBOXYLASE IN HUMAN POST-MORTEM BRAIN TISSUE FROM CONTROL AND HUNTINGTON''S CHOREA SUBJECTS

Abstract— GABA and its biosynthetic enzyme glutamic acid decarboxylase (GAD) remained remarkably stable for many hours after death in both human putamen obtained at autopsy and in mouse brain stored under conditions simulating the routine handling of human cadavers. GAD activity was profoundly influenced by agonal status in control but not in choreic subjects. Conversely, GABA concentrations were unaffected by the agonal status but showed a significant age-related decline. GAD activity and GABA concentrations were positively correlated in sudden death control cases but not in control cases suffering a protracted terminal illness or in choreic subjects. In choreic putamen there was an approximate 50% reduction in GABA concentration and GAD activity (correcting for agonal status) consistent with the hypothesis that striatal GABA-containing neurones degenerate in this disease. Since GABA concentrations are unaffected by agonal factors they may provide a reliable marker for the integrity of GABA systems provided that control and pathological groups are matched for age and delay in post-mortem sampling.     

Brain banks: benefits,limitations and cautions concerning the use of post-mortem brain tissue for molecular studies

Brain banks are facilities providing an interface between generous donation of nervous tissues and research laboratories devoted to increase our understanding of the diseases of the nervous system, discover new diagnostic targets, and develop new strategies. Considering this crucial role, it is important to learn about the suitabilities, limitations and proper handling of individual brain samples for particular studies. Several factors may interfere with preservation of DNA, RNA, proteins and lipids, and, therefore, special care must be taken first to detect sub-optimally preserved tissues and second to provide adequate material for each specific purpose. Basic aspects related with DNA, RNA and protein preservation include agonal state, post-mortem delay, temperature of storage and procedures of tissue preservation. Examination of DNA and RNA preservation is best done by using bioanalyzer technologies instead of less sensitive methods such as agarose gels. Adequate RNA preservation is mandatory in RNA microarray studies and adequate controls are necessary for proper PCR validation. Like for RNA, the preservation of proteins is not homogeneous since some molecules are more vulnerable than others. This aspect is crucial in the study of proteins including expression levels and possible post-translational modifications. Similarly, the reliability of functional and enzymatic studies in human post-mortem brain largely depends on protein preservation. Much less is known about other aspects, such as the effects of putative deleterious factors on epigenetic events such as methylation of CpGs in gene promoters, nucleosome preservation, histone modifications, and conservation of microRNA species. Most brains are appropriate for morphological approaches but not all brains are useful for certain biochemical and molecular studies.     

Tryptophan is a marker of human postmortem brain tissue quality

Postmortem human brain tissue is widely used in neuroscience research, but use of tissue originating from different brain bank centers is considered inaccurate because of possible heterogeneity in sample quality. There is thus a need for well-characterized markers to assess the quality of postmortem brain tissue. Toward this aim, we determined tryptophan (TRP) concentrations, phosphofructokinase-1 and glutamate decarboxylase activities in 119 brain tissue samples. These neurochemical parameters were tested in samples from autopsied individuals, including control and pathological cases provided by 10 different brain bank centers. Parameters were assessed for correlation with agonal state, postmortem interval, age and gender, brain region, preservation and freezing methods, storage conditions and storage time, RNA integrity, and tissue pH value. TRP concentrations were elevated significantly ( p  = 0.045) with increased postmortem interval; which might indicate increased protein degradation. Therefore, TRP concentration might be one useful and convenient marker for estimating the quality of human postmortem brain tissue.     

RNA integrity and the effect on the real-time qRT-PCR performance

The assessment of RNA integrity is a critical first step in obtaining meaningful gene expression data. Working with low-quality RNA may strongly compromise the experimental results of downstream applications which are often labour-intensive, time-consuming, and highly expensive. Using intact RNA is a key element for the successful application of modern molecular biological methods, like qRT-PCR or micro-array analysis. To verify RNA quality nowadays commercially available automated capillary-electrophoresis systems are available which are on the way to become the standard in RNA quality assessment. Profiles generated yield information on RNA concentration, allow a visual inspection of RNA integrity, and generate approximated ratios between the mass of ribosomal sub-units. In this review, the importance of RNA quality for the qRT-PCR was analyzed by determining the RNA quality of different bovine tissues and cell culture. Independent analysis systems are described and compared (OD measurement, NanoDrop, Bioanalyzer 2100 and Experion). Advantage and disadvantages of RNA quantity and quality assessment are shown in performed applications of various tissues and cell cultures. Further the comparison and correlation between the total RNA integrity on PCR performance as well as on PCR efficiency is described. On the basis of the derived results we can argue that qRT-PCR performance is affected by the RNA integrity and PCR efficiency in general is not affected by the RNA integrity. We can recommend a RIN higher than five as good total RNA quality and higher than eight as perfect total RNA for downstream application.     

[3H]Nipecotic Acid Binding to γ-Aminobutyric Acid Uptake Sites in Postmortem Human Brain

Binding of [3H]nipecotic acid, a proposed marker for GABAergic neurons, was investigated in postmortem human brain by use of a centrifugation assay. Binding was displaceable, apparently saturable, and to a single site, with typical KD and Bmax values of 1.85 microM and 124.2 pmol/mg of protein in the hippocampus. Regional distribution studies indicated a heterogeneous population of [3H]nipecotic acid binding sites with highest concentrations in the lateral globus pallidus. Putamen tissue from four cases of Huntington's disease showed a marked reduction in [3H]nipecotic acid binding. Binding correlated with both age and postmortem delay in the hippocampus. There was an effect of agonal state in which prolonged illness before death apparently caused a reduction in binding. Our results indicate that [3H]nipecotic acid may be used successfully as a marker for neuronal GABAergic uptake sites in human brain, but that the effects of variables such as age, postmortem delay, and agonal state must always be taken into account.     

Integrating protein structures and precomputed genealogies in the Magnum database: Examples with cellular retinoid binding proteins

Background

Gene expression microarray experiments are expensive to conduct and guidelines for acceptable quality control at intermediate steps before and after the samples are hybridised to chips are vague. We conducted an experiment hybridising RNA from human brain to 117 U133A Affymetrix GeneChips and used these data to explore the relationship between 4 pre-chip variables and 22 post-chip outcomes and quality control measures.

Results

We found that the pre-chip variables were significantly correlated with each other but that this correlation was strongest between measures of RNA quality and cRNA yield. Post-mortem interval was negatively correlated with these variables. Four principal components, reflecting array outliers, array adjustment, hybridisation noise and RNA integrity, explain about 75% of the total post-chip measure variability. Two significant canonical correlations existed between the pre-chip and post-chip variables, derived from MAS 5.0, dChip and the Bioconductor packages affy and affyPLM. The strongest (CANCOR 0.838, p < 0.0001) correlated RNA integrity and yield with post chip quality control (QC) measures indexing 3'/5' RNA ratios, bias or scaling of the chip and scaling of the variability of the signal across the chip. Post-mortem interval was relatively unimportant. We also found that the RNA integrity number (RIN) could be moderately well predicted by post-chip measures B_ACTIN35, GAPDH35 and SF.

Conclusion

We have found that the post-chip variables having the strongest association with quantities measurable before hybridisation are those reflecting RNA integrity. Other aspects of quality, such as noise measures (reflecting the execution of the assay) or measures reflecting data quality (outlier status and array adjustment variables) are not well predicted by the variables we were able to determine ahead of time. There could be other variables measurable pre-hybridisation which may be better associated with expression data quality measures. Uncovering such connections could create savings on costly microarray experiments by eliminating poor samples before hybridisation.     

Quantitative expression profile of distinct functional regions in the adult mouse brain

The adult mammalian brain is composed of distinct regions with specialized roles including regulation of circadian clocks, feeding, sleep/awake, and seasonal rhythms. To find quantitative differences of expression among such various brain regions, we conducted the BrainStars (B*) project, in which we profiled the genome-wide expression of ～50 small brain regions, including sensory centers, and centers for motion, time, memory, fear, and feeding. To avoid confounds from temporal differences in gene expression, we sampled each region every 4 hours for 24 hours, and pooled the samples for DNA-microarray assays. Therefore, we focused on spatial differences in gene expression. We used informatics to identify candidate genes with expression changes showing high or low expression in specific regions. We also identified candidate genes with stable expression across brain regions that can be used as new internal control genes, and ligand-receptor interactions of neurohormones and neurotransmitters. Through these analyses, we found 8,159 multi-state genes, 2,212 regional marker gene candidates for 44 small brain regions, 915 internal control gene candidates, and 23,864 inferred ligand-receptor interactions. We also found that these sets include well-known genes as well as novel candidate genes that might be related to specific functions in brain regions. We used our findings to develop an integrated database (http://brainstars.org/) for exploring genome-wide expression in the adult mouse brain, and have made this database openly accessible. These new resources will help accelerate the functional analysis of the mammalian brain and the elucidation of its regulatory network systems.     

GABA(A) receptor alpha-subunit proteins in human chronic alcoholics

Antibodies were raised against specific peptides from N-terminal regions of the alpha1 and alpha3 isoforms of the GABA(A) receptor, and used to assess the relative expression of these proteins in the superior frontal and primary motor cortices of 10 control, nine uncomplicated alcoholic and six cirrhotic alcoholic cases were matched for age and post-mortem delay. The regression of expression on post-mortem delay was not statistically significant for either isoform in either region. In both cortical areas, the regression of alpha1 expression on age differed significantly between alcoholic cases, which showed a decrease, and normal controls, which did not. Age had no effect on alpha3 expression. The alpha1 and alpha3 isoforms were found to be expressed differentially across cortical regions and showed a tendency to be expressed differentially across case groups. In cirrhotic alcoholics, alpha1 expression was greater in superior frontal than in motor cortex, whereas this regional difference was not significant in controls or uncomplicated alcoholics. In uncomplicated alcoholics, alpha3 expression was significantly lower in superior frontal than in motor cortex. Expression of alpha1 was significantly different from that of alpha3 in the superior frontal cortex of alcoholics, but not in controls. In motor cortex, there were no significant differences in expression between the isoforms in any case group.     

Expression Profiling with Oligonucleotide Arrays: Technologies and Applications for Neurobiology

DNA microarrays have been used in applications ranging from the assignment of gene function to analytical uses in prognostics. However, the detection sensitivity, cross hybridization, and reproducibility of these arrays can affect experimental design and data interpretation. Moreover, several technologies are available for fabrication of oligonucleotide microarrays. We review these technologies and performance attributes and, with data sets generated from human brain RNA, present statistical tools and methods to analyze data quality and to mine and visualize the data. Our data show high reproducibility and should allow an investigator to discern biological and regional variability from differential expression. Although we have used brain RNA as a model system to illustrate some of these points, the oligonucleotide arrays and methods employed in this study can be used with cell lines, tissue sections, blood, and other fluids. To further demonstrate this point, we provide data generated from total RNA sample sizes of 200 ng.