RESUMO
Gene expression analyses based on messenger RNA (mRNA) profiling require accurate data normalisation. When using endogenous reference genes, these have to be validated carefully. Therefore, we examined the transcript stability of 10 potential reference genes using quantitative real-time polymerase chain reaction: beta actin, 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase, TATA box-binding protein, hypoxanthine phosphoribosyl-transferase I, beta-2-microglobulin, hydroxymethylbilane synthase, succinate dehydrogenase complex, subunit A, cyclophilin A and ubiquitin C. The aim of the current study was to assess which reference genes show stable mRNA levels in human post mortem cardiac muscle, skeletal muscle and brain tissue. Considering cardiac muscle tissue, CYCA and TBP were identified as the most stable while in skeletal muscle tissue, SDHA and TBP, and in brain tissue, SDHA and HMBS turned out to be the most stable. Furthermore, we recommend a minimum of four carefully validated endogenous control genes for reliable data normalisation in human post mortem tissue. Parameters influencing the stability of transcript amounts were found to be mainly the post mortem interval in cardiac muscle and skeletal muscle tissue and the donor's cause of death in skeletal muscle and brain samples. Further parameters like gender, age at death and body mass index were found to influence mRNA quantities in skeletal muscle only. The set of stable control genes identified in this study may be used in further studies if the composition of the samples is similar to the one used here.
Assuntos
Encéfalo/metabolismo , Perfilação da Expressão Gênica , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , RNA Mensageiro/análise , Actinas/genética , Adulto , Fatores Etários , Índice de Massa Corporal , Causas de Morte , Ciclofilina A/genética , Feminino , Genética Forense , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Hidroximetilbilano Sintase/genética , Hipoxantina Fosforribosiltransferase/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Mudanças Depois da Morte , RNA Ribossômico 18S/genética , Succinato Desidrogenase/genética , Proteína de Ligação a TATA-Box/genética , Ubiquitina C/genética , Microglobulina beta-2/genéticaRESUMO
Single-nucleotide extension is a widespread method for typing Y-chromosomal single-nucleotide polymorphisms. In our study, we validated a multiplex minisequencing assay in a reduced-volume and in a low-volume approach. A four-plex assay was performed in a 6-microL multiplex reaction in 96-well microtiter reaction plates, which can be directly used for capillary electrophoresis. In a second approach, a six-plex assay was performed on a chemically structured glass slide. Both techniques have proven to be highly sensitive as well as time- and cost-saving, which makes them a valuable option not only for forensic purposes but also for population genetic studies where large sample numbers have to be analyzed. In the present paper, both techniques are compared and applied to analyze a population sample from the area of Turku, Finland. The most common haplogroup was found to be N1c*, which is nearly absent in western and central European populations. Additionally, 11 short tandem repeat markers were analyzed to further discriminate Y-chromosomal lineages.
Assuntos
Cromossomos Humanos Y , Impressões Digitais de DNA/métodos , Haplótipos , Polimorfismo de Nucleotídeo Único , Finlândia , Humanos , Masculino , Reação em Cadeia da Polimerase , Sequências de Repetição em TandemRESUMO
The aim of the present study was to examine an ultrasound-accelerated fixation technique that reduces the exposure time of the tissue to formaldehyde with respect to the analysis of nucleic acids. We extracted and analysed DNA and RNA from three series of autopsy specimens from five routine cases. Two series were shortly fixed in 4% buffered formalin (15 and 30 min, respectively) whilst being irradiated with high-frequency, high-intensity ultrasound. The last series (control) was routinely fixed in 4% buffered formalin for 24-48 h without irradiation. Although sufficient amounts of DNA of good quality could be extracted and amplified from all three series, the peak heights obtained from conventional fixation were smaller and allele dropout occurred more often, especially for the longer amplicons. RNA yield depended on the fixation procedure, i.e. the shortest fixation time led to the highest RNA yield and quality. No differences were observed with regard to the quality of the histological slides both with conventional and immunohistochemical staining methods. Keeping in mind the increasing need for molecular diagnosis, this fixation technique can be useful to ensure stable quality of nucleic acids in archived autopsy specimens.
Assuntos
DNA/análise , Fixadores , Formaldeído , RNA/análise , Fixação de Tecidos/métodos , Ultrassom , Encéfalo/patologia , Medicina Legal , Humanos , Rim/patologia , Fígado/patologia , Pulmão/patologia , Miocárdio/patologia , Pâncreas/patologia , Manejo de Espécimes , Baço/patologiaRESUMO
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.
Assuntos
DNA Mitocondrial/genética , Linfócitos/fisiologia , Adulto , Primers do DNA , Citometria de Fluxo , Humanos , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Relative quantification of mRNA using quantitative real-time reverse transcription (RT)-PCR is a commonly used method for analysis and comparison of gene expression levels. This method requires a normalisation of data against expression levels of a control gene. In the past, several ubiquitously expressed genes were used as such endogenous controls. When working with human tissue samples obtained during autopsy one has to deal with postmortem intervals of usually more than 10 h. The aim of this study was to investigate whether commonly used endogenous control genes show stability over various postmortem intervals. For this purpose, RNA was extracted from three different human tissues of five postmortem intervals ranging from 15 to 118 h. The Ct values from five commonly used endogenous control genes--beta-actin, B2M, CyPA, TBP, and UBC--were obtained by real-time RT-PCR. Results revealed a relatively high stability of Ct values in skeletal muscle tissue regarding different postmortem intervals. In heart and brain tissues, all endogenous controls were found to be highly variable. B2M appeared to be the least unstable control in this set. Nevertheless, all endogenous controls showed variability in their expression levels regarding both the stability among different tissues and different postmortem intervals. Data obtained in the present study show that postmortem mRNA degradation is a complex process, and that the use of one single endogenous control in gene expression studies of postmortem tissue would lead to erroneous data interpretation. Further studies on this topic should be performed in the future including an increased number of well documented samples.
Assuntos
Genética Forense/métodos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Autopsia , Química Encefálica , DNA Complementar/genética , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/química , Miocárdio/química , RNA Mensageiro/isolamento & purificaçãoRESUMO
Sequence analysis of the human mitochondrial genome (mtDNA) has proven to be a valuable tool in forensic identity testing and the analysis of crime scene stains. In contrast to the very expensive sequencing technique, typing of different length variants can greatly facilitate screening of a large number of traces for their relevance during casework. Within the mitochondrial control region, a dinucleotide (CA)( n ) repeat locus is present. To assess the discrimination power of this marker, we have determined (CA)( n ) allele distribution and the frequency of heteroplasmy in a population sample of 2,458 Germans. The inclination to develop heteroplasmic mixtures (CA)( n )/(CA)( n-1) was positively correlated with the number of CA repeats in the mtDNA. In addition, we have studied the inheritance patterns of (CA)( n ) repeat sequence heteroplasmy in two pedigrees. In one pedigree, we also found a length heteroplasmy in the homopolymeric C-tract (nt 303-309). Our data show stable inheritance of heteroplasmy within the homopolymeric C-stretch, but rather unstable inheritance regarding the (CA)( n ) repeat locus.
Assuntos
DNA Mitocondrial/genética , Repetições de Dinucleotídeos , Padrões de Herança , Genética Forense , Genética Populacional , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Low volume (LV) amplification (1 microL) of nuclear DNA (nucDNA) on a chemically structured chip is an appropriate and highly sensitive method to simultaneously amplify amelogenin and 15 forensically relevant short tandem repeats (STR). In this study, a combined method using on-chip LV amplification of mitochondrial DNA (mtDNA) and subsequent on-chip LV cycle sequencing was established to obtain a method, which is sensitive and robust enough to allow reliable analysis of DNA amounts representing the single cell level. All the necessary steps of the procedure--except for the purification of the sequencing products--were accomplished within the same final 2-microL reaction volume.
Assuntos
Impressões Digitais de DNA/métodos , DNA Mitocondrial/análise , Eletroforese em Microchip/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase/métodos , DNA Mitocondrial/genética , Humanos , Repetições de Microssatélites/genéticaRESUMO
Recently, several authors described the observation that RNA degradation does not correlate with the postmortem interval (PMI), but rather with other parameters like environmental impact and the circumstances of death. Therefore, the question arose if the analysis of gene expression could be a valuable tool in forensic genetics to contribute to the determination of the cause of death. In our study, six human tissues obtained from six individuals with PMI varying between 15 and 118 h were used for total RNA extraction. Quantification was performed using a GAPDH real-time assay, and the quality of mRNA was checked by amplification of different fragment lengths of the GAPDH transcript. In our set of samples, nearly all tissues in all PMI revealed satisfactory results, while skeletal muscle, followed by brain and heart, gave the best results. No correlation between PMI and RNA degradation could be detected, as very good results were observed for all tissues from the individual with the longest PMI. The highly promising results obtained in this study raise hopes that in the near future several fields of forensic investigation may profit from additional information about gene expression patterns and their correlation with pathological findings.
Assuntos
Autopsia/métodos , Causas de Morte , Perfilação da Expressão Gênica/métodos , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Gliceraldeído-3-Fosfato Desidrogenases , Humanos , Reação em Cadeia da Polimerase/métodos , Mudanças Depois da Morte , Estabilidade de RNA , Reprodutibilidade dos Testes , Fatores de Tempo , Distribuição TecidualRESUMO
A collaborative study was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the performance of Y-chromosome binary polymorphism analysis in different European laboratories. Four blood samples were sent to the laboratories, to be analysed for 11 Y-chromosome single nucleotide polymorphisms (SNPs): SRY-1532, M40, M35, M213, M9, 92R7, M17, P25, M18, M153 and M167. All the labs were also asked to submit a population study including these markers. All participating laboratories reported the same results, indicating the reproducibility and robustness of Y-chromosome SNP typing. A total of 535 samples from six different European populations were also analysed. In Galicia (NW Spain) and Belgium, the most frequent haplogroup was R1b*(xR1b1,R1b3df). Haplogroup F*(xK) is one of the most frequent in Austria and Denmark, while the lowest frequency appear in Belgium. Haplogroup frequencies found in this collaborative study were compared with previously published European Y-chromosome haplogroup data.
Assuntos
Cromossomos Humanos Y , Impressões Digitais de DNA , Genética Populacional , Polimorfismo de Nucleotídeo Único , Europa (Continente) , Marcadores Genéticos , Haplótipos , Humanos , Masculino , Reação em Cadeia da Polimerase , Reprodutibilidade dos TestesRESUMO
To test for human population substructure and to investigate human population history we have analysed Y-chromosome diversity using seven microsatellites (Y-STRs) and ten binary markers (Y-SNPs) in samples from eight regionally distributed populations from Poland (n = 913) and 11 from Germany (n = 1,215). Based on data from both Y-chromosome marker systems, which we found to be highly correlated (r = 0.96), and using spatial analysis of the molecular variance (SAMOVA), we revealed statistically significant support for two groups of populations: (1) all Polish populations and (2) all German populations. By means of analysis of the molecular variance (AMOVA) we observed a large and statistically significant proportion of 14% (for Y-SNPs) and 15% (for Y-STRs) of the respective total genetic variation being explained between both countries. The same population differentiation was detected using Monmonier's algorithm, with a resulting genetic border between Poland and Germany that closely resembles the course of the political border between both countries. The observed genetic differentiation was mainly, but not exclusively, due to the frequency distribution of two Y-SNP haplogroups and their associated Y-STR haplotypes: R1a1*, most frequent in Poland, and R1*(xR1a1), most frequent in Germany. We suggest here that the pronounced population differentiation between the two geographically neighbouring countries, Poland and Germany, is the consequence of very recent events in human population history, namely the forced human resettlement of many millions of Germans and Poles during and, especially, shortly after World War II. In addition, our findings have consequences for the forensic application of Y-chromosome markers, strongly supporting the implementation of population substructure into forensic Y chromosome databases, and also for genetic association studies.
Assuntos
Cromossomos Humanos Y/genética , Demografia , Variação Genética , Emigração e Imigração , Geografia , Alemanha , Haplótipos , Humanos , Masculino , Repetições de Microssatélites , Polônia , Polimorfismo de Nucleotídeo Único , II Guerra MundialRESUMO
We have observed three hitherto undescribed off-ladder alleles at three widely used STR loci. These were isolated, sequenced and designated as follows: allele 10 (D2S1338, one case), allele 21 (D3S1358, two cases) and allele 6.2 (D19S433, six cases). These sequences are described in comparison to non-variant alleles, and their implications for the semi-automated STR analysis will be discussed.
Assuntos
Alelos , Sequências de Repetição em Tandem/genética , Sequência de Bases , Humanos , Análise de Sequência de DNARESUMO
In the course of routine genotyping of forensic reference samples by multiplex PCR, an allelic drop-out due to mutations in the primer binding regions of the highly polymorphic STR marker ACTBP2 was observed in 17 samples. The variation rate was estimated to be 0.0014 (95% confidence interval: 0.0006-0.003). The most frequently found mutation was an G to A transition in the reverse primer binding region which was present in 14 out of 17 cases. To overcome the problem we have added a modified reverse primer to different multiplex kits that led to the correct genotype.
Assuntos
Impressões Digitais de DNA/métodos , Primers do DNA/genética , Perda de Heterozigosidade/genética , Sequências de Repetição em Tandem/genética , Sítios de Ligação , HumanosRESUMO
Okihiro syndrome refers to the association of forearm malformations with Duane syndrome of eye retraction. Based on the reported literature experience, clinical diagnosis of the syndrome can be elusive, owing to the variable presentation in families reported. Specifically, there is overlap of clinical features with other conditions, most notably Holt-Oram syndrome, a condition resulting from mutation of the TBX5 locus and Townes-Brocks syndrome, known to be caused by mutations in the SALL1 gene. Arising from our observation of several malformations in Okihiro syndrome patients which are also described in Townes-Brocks syndrome, we postulated that Okihiro syndrome might result from mutation of another member of the human SALL gene family. We have characterized the human SALL4 gene on chromosome 20q13.13-q13.2. Moreover, we have identified literature reports of forelimb malformations in patients with cytogenetically identifiable abnormalities of this region. We here present evidence in 5 of 8 affected families that mutation at this locus results in the Okihiro syndrome phenotype.