Urinary miRNAs: Technical Updates.

Due to its non-invasive nature and easy accessibility, urine serves as a convenient bi-ological fluid for research purposes. Furthermore, urine samples are uncomplicated to preserve and relatively inexpensive. MicroRNA (miRNAs), small molecules that regulate gene expression post-transcriptionally, play vital roles in numerous cellular processes, including apoptosis, cell differentiation, development, and proliferation. Their dysregulated expression in urine has been proposed as a potential bi-omarker for various human diseases, including bladder cancer. To draw reliable conclusions about the roles of urinary miRNAs in human diseases, it is essential to have dependable and reproducible methods for miRNA extraction and profiling. In this review, we address the technical challenges associated with studying urinary miRNAs and provide an update on the current technologies used for urinary miRNA isolation, quality control assessment, and miRNA profiling, highlighting both their advantages and limitations.

Human sample authentication in biomedical research: comparison of two platforms.

Samples used in biomedical research are often collected over years, in some cases from subjects that may have died and thus cannot be retrieved in any way. The value of these samples is priceless. Sample misidentification or mix-up are unfortunately common problems in biomedical research and can eventually result in the publication of incorrect data. Here we have compared the Fluidigm SNPtrace and the Agena iPLEX Sample ID panels for the authentication of human genomic DNA samples. We have tested 14 pure samples and simulated their cross-contamination at different percentages (2%, 5%, 10%, 25% and 50%). For both panels, we report call rate, allele intensity/probability score, performance in distinguishing pure samples and contaminated samples at different percentages, and sex typing. We show that both panels are reliable and efficient methods for sample authentication and we highlight their advantages and disadvantages. We believe that the data provided here is useful for sample authentication especially in biorepositories and core facility settings.

Bioarchaeology-related studies in the Arabian Gulf: potentialities and shortcomings.

Archaeological studies provide a powerful tool to understand the prehistoric societies, especially when combined to cutting-edge morphological and molecular anthropological analyses, allowing reconstructing past population dynamics, admixture events, and socio-cultural changes. Despite the advances achieved in the last decades by archaeological studies worldwide, several regions of the World have been spared from this scientific improvement due to various reasons. The Arabian Gulf represents a unique ground to investigate, being the passageway for human migrations and one of the hypothesized areas in which Neanderthal introgression occurred. A number of archaeological sites are currently present in the Arabian Gulf and have witnessed the antiquity and the intensiveness of the human settlements in the region. Nevertheless, the archaeological and anthropological investigation in the Gulf is still in its infancy. Data collected through archaeological studies in the area have the potential to help answering adamant questions of human history from the beginning of the structuring of genetic diversity in human species to the Neolithisation process. This review aims at providing an overview of the archaeological studies in the Arabian Gulf with special focus to Qatar, highlighting potentialities and shortcomings.

Differential responsiveness to BRAF inhibitors of melanoma cell lines BRAF V600E-mutated.

BACKGROUND: Most mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution. Patient management is often based on the use of specific inhibitors targeting this mutation. METHODS: DNA and RNA mutation status was assessed in 15 melanoma cell lines by Sanger sequencing and RNA-seq. We tested the cell lines responsiveness to BRAF inhibitors (vemurafenib and PLX4720, BRAF-specific and sorafenib, BRAF non-specific). Cell proliferation was assessed by MTT colorimetric assay. BRAF V600E RNA expression was assessed by qPCR. Expression level of phosphorylated-ERK protein was assessed by Western Blotting as marker of BRAF activation. RESULTS: Three cell lines were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that they express low level of BRAF RNA and the expression may be in favor of the WT allele. We tested whether the discordant cell lines responded differently to BRAF-specific inhibitors. Their proliferation rate decreased after treatment with vemurafenib and PLX4720 but was not affected by sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the control. Western Blot analysis revealed a decreased expression of p-ERK protein in the BRAF V600E control cell line and in the discordant cell lines upon treatment with BRAF-specific inhibitors. The discordant cell lines showed a lower responsiveness to BRAF inhibitors when compared to the BRAF V600E control cell line. The results obtained from the inhibition experiment and molecular analyses were also confirmed in three additional cell lines. CONCLUSION: Cell lines carrying V600E mutation at the DNA level may respond differently to BRAF targeted treatment potentially due to a lower V600E RNA expression.

Influence of storage conditions of small volumes of blood on immune transcriptomic profiles.

OBJECTIVE: Transcriptome analysis of human whole blood is used to discover biomarkers of diseases and to assess phenotypic traits. Here we have collected small volumes of blood in Tempus solution and tested whether different storage conditions have an impact on transcriptomic profiling. Fifty µl of blood were collected in 100µl of Tempus solutions, freezed at - 20 °C for 1 day and eventually thawed, stored and processed under five different conditions: (i) - 20 °C for 1 week; (ii) +4 °C for 1 week; (iii) room temperature for 1 week; (iv) room temperature for 1 day, - 20 °C for 1 day, room temperature until testing at day 7, (v) - 20 °C for 1 week, RNA was isolated and stored in GenTegra solution. We used 272 immune transcript specific assays to test the expression profiling using qPCR based Fluidigm BioMark HD dynamic array. RESULTS: RNA yield ranged between 0.17 and 1.39µg. Except for one sample, RIN values were > 7. Using Principal Component Analysis, we saw that the storage conditions did not drive sample distribution. The condition that showed larger variability was the RT-FR-RT (room temperature-freezing-room temperature), suggesting that freezing-thawing cycles may have a worse effect on data reproducibility than keeping the samples at room temperature.

Updates on the Current Technologies for microRNA Profiling.

MicroRNAs are RNA molecules of ~22 nt length that regulate gene expression posttranscriptionally. The role of miRNAs has been reported in many cellular processes including apoptosis, cell differentiation, development and proliferation. The dysregulated expression of miRNAs has been proposed as a biomarker for the diagnosis, onset and prognosis of human diseases. The utility of miRNA profiles to identify and discriminate patients from healthy individuals is highly dependent on the sensitivity and specificity of the technologies used for their detection and the quantity and quality of starting material. In this review, we present an update of the current technologies for the extraction, QC assessment and detection of miRNAs with special focus to the most recent methods, discussing their advantages as well as their shortcomings.

Evaluation of Methods for the Extraction of Microbial DNA From Vaginal Swabs Used for Microbiome Studies.

Background: The composition of the microbiome in human body sites plays an important role in health. The vaginal environment is colonized by several species of bacteria that have a major influence on reproductive health. The advancement of sequencing technologies has made the assessment of the composition of the microbiota possible through microbial DNA extraction and sequencing. Therefore, it is of a paramount importance to select a sensitive and reproducible DNA extraction method, that facilitates isolation of microbial DNA with a sufficient quantity and purity, from microbial species living in the vaginal environment. Here, we have evaluated four different DNA extraction protocols from self-collected vaginal swabs. Methods: Five healthy female volunteers were enrolled in the study. Each donor was asked to self-collect 4 samples using Copan ESwab. DNA was extracted using Qiagen DNeasy kit and three modified protocols of the MoBio PowerSoil kit ("DNeasy PowerSoil" after acquisition from Qiagen). DNA quantity and integrity was checked through Nanodrop and LabChip GX. DNA was further tested through quantitative real-time PCR (qPCR) and 16S sequencing. Vaginal microbiota diversities were determined using MiSeq-Illumina high-throughput sequencing of bacterial 16S rDNA V1-V3 fingerprint. Sequencing data were analyzed using QIIME pipeline. Results: Qiagen DNeasy protocol resulted in the highest DNA yield as compared to the modified protocols of MoBio Powersoil kit. The size of the DNA extracted using each protocol was ~40 kb. Qiagen DNeasy protocol gave the highest Genomic Quality Score (average ± standard deviation: 4.24 ± 0.36), followed by the different MoBio Powersoil protocols. A substantial variability in microbial DNA abundance was found across the protocols. The vaginal microbiota of the healthy volunteers was dominated by Lactobacillus species. MoBio Powersoil kit provided a significantly higher alpha diversity as compared to the Qiagen DNeasy kit, while beta diversity measures did not reveal any significant cluster changes, except when the Bray-Curtis method was applied. Conclusion: We were able to isolate microbial DNA from the vaginal swabs. Qiagen DNeasy method gave the highest DNA yield and quality but was not optimal in detecting microbial diversity. The modified MoBio PowerSoil protocols showed higher microbial diversities as compared to the standard protocol.

Monoallelic expression in melanoma.

BACKGROUND: Monoallelic expression (MAE) is a frequent genomic phenomenon in normal tissues, however its role in cancer is yet to be fully understood. MAE is defined as the expression of a gene that is restricted to one allele in the presence of a diploid heterozygous genome. Constitutive MAE occurs for imprinted genes, odorant receptors and random X inactivation. Several studies in normal tissues have showed MAE in approximately 5-20% of the cases. However, little information exists on the MAE rate in cancer. In this study we assessed the presence and rate of MAE in melanoma. The genetic basis of melanoma has been studied in depth over the past decades, leading to the identification of mutations/genetic alterations responsible for melanoma development. METHODS: To examine the role of MAE in melanoma we used 15 melanoma cell lines and compared their RNA-seq data with genotyping data obtained by the parental TIL (tumor infiltrating lymphocytes). Genotyping was performed using the Illumina HumanOmni1 beadchip. The RNA-seq library preparation and sequencing was performed using the Illumina TruSeq Stranded Total RNA Human Kit and subsequently sequenced using a HiSeq 2500 according to manufacturer's guidelines. By comparing genotyping data with RNA-seq data, we identified SNPs in which DNA genotypes were heterozygous and corresponding RNA genotypes were homozygous. All homozygous DNA genotypes were removed prior to the analysis. To confirm the validity to detect MAE, we examined heterozygous DNA genotypes from X chromosome of female samples as well as for imprinted and olfactory receptor genes and confirmed MAE. RESULTS: MAE was detected in all 15 cell lines although to a different rate. When looking at the B-allele frequencies we found a preferential pattern of complete monoallelic expression rather then differential monoallelic expression across the 15 melanoma cell lines. As some samples showed high differences in the homozygous and heterozygous call rate, we looked at the single chromosomes and showed that MAE may be explained by underlying large copy number imbalances in some instances. Interestingly these regions included genes known to play a role in melanoma initiation and progression. Nevertheless, some chromosome regions showed MAE without CN imbalances suggesting that additional mechanisms (including epigenetic silencing) may explain MAE in melanoma. CONCLUSION: The biological implications of MAE are yet to be realized. Nevertheless, our findings suggest that MAE is a common phenomenon in melanoma cell lines. Further analyses are currently being undertaken to evaluate whether MAE is gene/pathway specific and to understand whether MAE can be employed by cancers to achieve a more aggressive phenotype.

The immune-related role of BRAF in melanoma.

BACKGROUND: The existence of a dichotomy between immunologically active and quiescent tumor phenotypes has been recently recognized in several types of cancer. The activation of a Th1 type of immune signature has been shown to confer better prognosis and likelihood to respond to immunotherapy. However, whether such dichotomy depends on the genetic make-up of individual cancers is not known yet. BRAF and NRAS mutations are commonly acquired during melanoma progression. Here we explored the role of BRAF and NRAS mutations in influencing the immune phenotype based on a classification previously identified by our group. METHODS: One-hundred-thirteen melanoma metastases underwent microarray analysis and BRAF and NRAS genotyping. Allele-specific PCR was also performed in order to exclude low-frequency mutations. RESULTS: Comparison between BRAF and NRAS mutant versus wild type samples identified mostly constituents or regulators of MAPK and related pathways. When testing gene lists discriminative of BRAF, NRAS and MAPK alterations, we found that 112 BRAF-specific transcripts were able to distinguish the two immune-related phenotypes already described in melanoma, with the poor phenotype associated mostly with BRAF mutation. Noteworthy, such association was stronger in samples displaying low BRAF mRNA expression. However, when testing NRAS mutations, we were not able to find the same association. CONCLUSION: This study suggests that BRAF mutation-related specific transcripts associate with a poor phenotype in melanoma and provide a nest for further investigation.