Small RNA-sequencing (RNA-Seq) is being increasingly used for profiling of circulating microRNAs ... more Small RNA-sequencing (RNA-Seq) is being increasingly used for profiling of circulating microRNAs (miRNAs), a new group of promising biomarkers. Unfortunately, small RNA-Seq protocols are prone to biases limiting quantification accuracy, which motivated development of several novel methods. Here, we present comparison of all small RNA-Seq library preparation approaches that are commercially available for quantification of miRNAs in biofluids. Using synthetic and human plasma samples, we compared performance of traditional two-adaptor ligation protocols (Lexogen, Norgen) as well as methods using randomized adaptors (NEXTflex), polyadenylation (SMARTer), circularization (RealSeq), capture probes (EdgeSeq) or unique molecular identifiers, UMIs (QIAseq). Globally, there was no single protocol outperforming others across all metrics. We documented limited overlap of measured miRNA profiles between methods largely owing to protocol-specific biases. We found that methods designed to minimiz...
MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in m... more MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches t...
Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information fo... more Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimen...
Background: Among emerging circulating biomarkers, miRNA has the potential to detect lung cancer ... more Background: Among emerging circulating biomarkers, miRNA has the potential to detect lung cancer and follow the course of the disease. However, miRNA analysis deserves further standardization before implementation into clinical trials or practice. Here, we performed international ring experiments to explore (pre)-analytical factors relevant to the outcome of miRNA blood tests in the context of the EU network CANCER-ID. Methods: Cell-free (cfmiRNA) and extracellular vesicle-derived miRNA (EVmiRNA) were extracted using the miRNeasy Serum/Plasma Advanced, and the ExoRNeasy Maxi kit, respectively, in a plasma cohort of 27 NSCLC patients and 20 healthy individuals. Extracted miRNA was investigated using small RNA sequencing and hybridization platforms. Validation of the identified miRNA candidates was performed using quantitative PCR. Results: We demonstrate the highest read counts in healthy individuals and NSCLC patients using QIAseq. Moreover, QIAseq showed 15.9% and 162.9% more cfmiR...
BACKGROUND In human body fluids, microRNA (miRNA) can be found as circulating cell-free miRNA (cf... more BACKGROUND In human body fluids, microRNA (miRNA) can be found as circulating cell-free miRNA (cfmiRNA), as well as secreted into extracellular vesicles (EVmiRNA). miRNAs are being intensively evaluated as minimally invasive liquid biopsy biomarkers in patients with cancer. The growing interest in developing clinical assays for circulating miRNA necessitates careful consideration of confounding effects of preanalytical and analytical parameters. METHODS By using reverse transcription quantitative real-time PCR and next-generation sequencing (NGS), we compared extraction efficiencies of 5 different protocols for cfmiRNA and 2 protocols for EVmiRNA isolation in a multicentric manner. The efficiency of the different extraction methods was evaluated by measuring exogenously spiked cel-miR-39 and 6 targeted miRNAs in plasma from 20 healthy individuals. RESULTS There were significant differences between the tested methods. Although column-based extraction methods were highly effective for...
Asymmetric cell division is a ubiquitous feature during the development of higher organisms. Asym... more Asymmetric cell division is a ubiquitous feature during the development of higher organisms. Asymmetry is achieved by differential localization or activities of biological molecules such as proteins, and coding and non-coding RNAs. Here, we present subcellular transcriptomic and proteomic analyses along the animal-vegetal axis of Xenopus laevis eggs. More than 98% of the maternal mRNAs could be categorized into four localization profile groups: animal, vegetal, extremely vegetal, and a newly described group of mRNAs that we call extremely animal, which are mRNAs enriched in the animal cortex region. 3'UTRs of localized mRNAs were analyzed for localization motifs. Several putative motifs were discovered for vegetal and extremely vegetal mRNAs, while no distinct conserved motifs for the extremely animal mRNAs were identified, suggesting different localization mechanisms. Asymmetric profiles were also found for proteins, with correlation to those of corresponding mRNAs. Based on un...
The aim of this study was to elucidate the molecular status of single human adult germ stem cells... more The aim of this study was to elucidate the molecular status of single human adult germ stem cells (haGSCs) and haGSC colonies, which spontaneously developed from the CD49f MACS and matrix- (collagen-/laminin+ binding-) selected fraction of enriched spermatogonia. Single-cell transcriptional profiling by Fluidigm BioMark system of a long-term cultured haGSCs cluster in comparison to human embryonic stem cells (hESCs) and human fibroblasts (hFibs) revealed that haGSCs showed a characteristic germ- and pluripotency-associated gene expression profile with some similarities to hESCs and with a significant distinction from somatic hFibs. Genome-wide comparisons with microarray analysis confirmed that different haGSC colonies exhibited gene expression heterogeneity with more or less pluripotency. The results of this study confirm that haGSCs are adult stem cells with a specific molecular gene expression profile in vitro, related but not identical to true pluripotent stem cells. Under ES-ce...
MicroRNAs are a family of naturally occurring small noncoding RNA molecules that play an importan... more MicroRNAs are a family of naturally occurring small noncoding RNA molecules that play an important regulatory role in gene expression. They are suggested to regulate a large proportion of protein encoding genes by mediating the translational suppression and posttranscriptional control of gene expression. Recent findings show that microRNAs are emerging as important regulators of cellular differentiation and dedifferentiation, and are deeply involved in developmental processes including human preimplantation development. They keep a balance between pluripotency and differentiation in the embryo and embryonic stem cells. Moreover, it became evident that dysregulation of microRNA expression may play a fundamental role in progression and dissemination of different cancers including ovarian cancer. The interest is still increased by the discovery of exosomes, that is, cell-derived vesicles, which can carry different proteins but also microRNAs between different cells and are involved in ...
The advent of molecular characterization of tissues has brought an increasing emphasis on the qua... more The advent of molecular characterization of tissues has brought an increasing emphasis on the quality of biospecimens, starting with the tissue procurement process. RNA levels are particularly affected by factors in the collection process, but the influence of different pre-analytical factors is not well understood. Here we present the influence of tissue specimen size, as well as the transport and freezing protocols, on RNA quality. Large, medium, and smaller porcine liver samples were stored either dry, on moist gauze, or in salt solution for various times, and then frozen in either liquid nitrogen or in pre-cooled isopentane. Large and small human liver samples were frozen in pre-cooled isopentane either immediately or after one hour at room temperature. The small samples were stored dry, on moist gauze, or in salt solution. RNA was isolated and RIN values were measured. The RNA for six standard reference genes from human liver was analyzed by RT-qPCR, and tissue morphology was assessed for artifacts of freezing. Experiments using porcine liver samples showed that RNA derived from smaller samples was more degraded after one hour of cold ischemia, and that cooled transport is preferable. Human liver samples showed significant RNA degradation after 1 h of cold ischemia, which was more pronounced in smaller samples. RNA integrity was not significantly influenced by the transport or freezing method, but changes in gene expression were observed in samples either transported on gauze or in salt solution. Based on observations in liver samples, smaller samples are more subject to gene expression variability introduced by post-excision sample handling than are larger samples. Small biopsies should be transported on ice and snap frozen as soon as possible after acquisition from the patient.
We have examined the imprecision in the estimation of PCR efficiency by means of standard curves ... more We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1-16 qPCR replicates per concentration and we tested 2-10 l volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3-4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range.
We have measured the expression of 41 maternal mRNAs in individual blastomeres collected from the... more We have measured the expression of 41 maternal mRNAs in individual blastomeres collected from the 8 to 32-cell Xenopus laevis embryos to determine when and how asymmetry in the body plan is introduced. We demonstrate that the asymmetry along the animal-vegetal axis in the oocyte is transferred to the daughter cells during early cell divisions. All studied mRNAs are distributed evenly among the set of animal as well as vegetal blastomeres. We find no asymmetry in mRNA levels that might be ascribed to the dorso-ventral specification or the left-right axis formation. We hypothesize that while the animal-vegetal asymmetry is a consequence of mRNA gradients, the dorso-ventral and left-right axes specifications are induced by asymmetric distribution of other biomolecules, probably proteins.
The aim of this study was to trigger the expression of genes related to oocytes in putative ovari... more The aim of this study was to trigger the expression of genes related to oocytes in putative ovarian stem cells scraped from the ovarian surface epithelium of women with premature ovarian failure and cultured in vitro in the presence of follicular fluid, rich in substances for oocyte growth and maturation. Ovarian surface epithelium was scraped and cell cultures were set up by scrapings in five women with nonfunctional ovaries and with no naturally present mature follicles or oocytes. In the presence of donated follicular fluid putative stem cells grew and developed into primitive oocyte-like cells. A detailed single-cell gene expression profiling was performed to elucidate their genetic status in comparison to human embryonic stem cells, oocytes, and somatic fibroblasts. The ovarian cell cultures depleted/converted reproductive hormones from the culture medium. Estradiol alone or together with other substances may be involved in development of these primitive oocyte-like cells. The ...
BACKGROUND Determining the transcriptional profile of circulating tumor cells (CTCs) may allow th... more BACKGROUND Determining the transcriptional profile of circulating tumor cells (CTCs) may allow the acquisition of clinically relevant information while overcoming tumor heterogeneity-related biases associated with use of tissue samples for biomarker assessment. However, such molecular characterization is challenging because CTCs are rare and outnumbered by blood cells. METHODS Here, we describe a technical protocol to measure the expression of >29 000 genes in CTCs captured from whole blood with magnetic beads linked with antibodies against epithelial cell adhesion molecule (EpCAM) and the carcinoma-associated mucin, MUC1, designed to be used for CTC characterization in clinical samples. Low numbers of cells (5–200) from the MCF7 and MDA-MB-468 breast cancer cell lines were spiked in healthy donor blood samples and isolated with the AdnaTest EMT-1/Stem CellSelect kit. Gene expression profiles (GEPs) were obtained with the WG-DASL HT assay and compared with GEPs obtained from RNA ...
In healthy humans, 60–70% of the B lymphocytes produce kappa light chains, while the remaining ce... more In healthy humans, 60–70% of the B lymphocytes produce kappa light chains, while the remaining cells produce lambda light chains. Malignant transformation and clonal expansion of B lymphocytes lead to an altered kappa : lambda expression ratio, which is an important diagnostic criteria of lymphomas. Here, we compared three methods for clonality determination of suspected B cell lymphomas. Tumor biopsies from 55 patients with B cell malignancies, 5 B-lymphoid tumor cell lines, and 20 biopsies from patients with lymphadenitis were analyzed by immunohistochemistry, flow cytometry, and reverse transcription quantitative real-time PCR. Clonality was determined by immunohistochemistry in 52/53 cases, flow cytometry in 30/39 cases, and reverse transcription quantitative real-time PCR in 33/55 cases. In conclusion, immunohistochemistry was superior to flow cytometry and reverse transcription quantitative real-time PCR for clonality identification. Flow cytometry and reverse transcription qu...
There is an increasing need for proper quality control tools in the pre-analytical phase of the m... more There is an increasing need for proper quality control tools in the pre-analytical phase of the molecular diagnostic workflow. The aim of the present study was to identify biomarkers for monitoring pre-analytical mRNA quality variations in two different types of blood collection tubes, K2EDTA (EDTA) tubes and PAXgene Blood RNA Tubes (PAXgene tubes). These tubes are extensively used both in the diagnostic setting as well as for research biobank samples. Blood specimens collected in the two different blood collection tubes were stored for varying times at different temperatures, and microarray analysis was performed on resultant extracted RNA. A large set of potential mRNA quality biomarkers for monitoring post-phlebotomy gene expression changes and mRNA degradation in blood was identified. qPCR assays for the potential biomarkers and a set of relevant reference genes were generated and used to pre-validate a sub-set of the selected biomarkers. The assay precision of the potential qPC...
One purpose of the EC funded project, SPIDIA, is to develop evidence-based quality guidelines for... more One purpose of the EC funded project, SPIDIA, is to develop evidence-based quality guidelines for the pre-analytical handling of blood samples for RNA molecular testing. To this end, two pan-European External Quality Assessments (EQAs) were implemented. Here we report the results of the second SPIDIA-RNA EQA. This second study included modifications in the protocol related to the blood collection process, the shipping conditions and pre-analytical specimen handling for participants. Participating laboratories received two identical proficiency blood specimens collected in tubes with or without an RNA stabilizer. For pre-defined specimen storage times and temperatures, laboratories were asked to perform RNA extraction from whole blood according to their usual procedure and to return extracted RNA to the SPIDIA facility for further analysis. These RNA samples were evaluated for purity, yield, integrity, stability, presence of interfering substances, and gene expression levels for the ...
Real-time PCR tomography is a novel, quantitative method for measuring localized RNA expression p... more Real-time PCR tomography is a novel, quantitative method for measuring localized RNA expression profiles within single cells. We demonstrate its usefulness by dissecting an oocyte from Xenopus laevis into slices along its animal-vegetal axis, extracting its RNA and measuring the levels of 18 selected mRNAs by real-time RT-PCR. This identified two classes of mRNA, one preferentially located towards the animal, the other towards the vegetal pole. mRNAs within each group show comparable intracellular gradients, suggesting they are produced by similar mechanisms. The polarization is substantial, though not extreme, with around 5% of vegetal gene mRNA molecules detected at the animal pole, and around 50% of the molecules in the far most vegetal section. Most animal pole mRNAs were found in the second section from the animal pole and in the central section, which is where the nucleus is located. mRNA expression profiles did not change following in vitro fertilization and we conclude that the cortical rotation that follows fertilization has no detectable effect on intracellular mRNA gradients.
Small RNA-sequencing (RNA-Seq) is being increasingly used for profiling of circulating microRNAs ... more Small RNA-sequencing (RNA-Seq) is being increasingly used for profiling of circulating microRNAs (miRNAs), a new group of promising biomarkers. Unfortunately, small RNA-Seq protocols are prone to biases limiting quantification accuracy, which motivated development of several novel methods. Here, we present comparison of all small RNA-Seq library preparation approaches that are commercially available for quantification of miRNAs in biofluids. Using synthetic and human plasma samples, we compared performance of traditional two-adaptor ligation protocols (Lexogen, Norgen) as well as methods using randomized adaptors (NEXTflex), polyadenylation (SMARTer), circularization (RealSeq), capture probes (EdgeSeq) or unique molecular identifiers, UMIs (QIAseq). Globally, there was no single protocol outperforming others across all metrics. We documented limited overlap of measured miRNA profiles between methods largely owing to protocol-specific biases. We found that methods designed to minimiz...
MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in m... more MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches t...
Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information fo... more Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimen...
Background: Among emerging circulating biomarkers, miRNA has the potential to detect lung cancer ... more Background: Among emerging circulating biomarkers, miRNA has the potential to detect lung cancer and follow the course of the disease. However, miRNA analysis deserves further standardization before implementation into clinical trials or practice. Here, we performed international ring experiments to explore (pre)-analytical factors relevant to the outcome of miRNA blood tests in the context of the EU network CANCER-ID. Methods: Cell-free (cfmiRNA) and extracellular vesicle-derived miRNA (EVmiRNA) were extracted using the miRNeasy Serum/Plasma Advanced, and the ExoRNeasy Maxi kit, respectively, in a plasma cohort of 27 NSCLC patients and 20 healthy individuals. Extracted miRNA was investigated using small RNA sequencing and hybridization platforms. Validation of the identified miRNA candidates was performed using quantitative PCR. Results: We demonstrate the highest read counts in healthy individuals and NSCLC patients using QIAseq. Moreover, QIAseq showed 15.9% and 162.9% more cfmiR...
BACKGROUND In human body fluids, microRNA (miRNA) can be found as circulating cell-free miRNA (cf... more BACKGROUND In human body fluids, microRNA (miRNA) can be found as circulating cell-free miRNA (cfmiRNA), as well as secreted into extracellular vesicles (EVmiRNA). miRNAs are being intensively evaluated as minimally invasive liquid biopsy biomarkers in patients with cancer. The growing interest in developing clinical assays for circulating miRNA necessitates careful consideration of confounding effects of preanalytical and analytical parameters. METHODS By using reverse transcription quantitative real-time PCR and next-generation sequencing (NGS), we compared extraction efficiencies of 5 different protocols for cfmiRNA and 2 protocols for EVmiRNA isolation in a multicentric manner. The efficiency of the different extraction methods was evaluated by measuring exogenously spiked cel-miR-39 and 6 targeted miRNAs in plasma from 20 healthy individuals. RESULTS There were significant differences between the tested methods. Although column-based extraction methods were highly effective for...
Asymmetric cell division is a ubiquitous feature during the development of higher organisms. Asym... more Asymmetric cell division is a ubiquitous feature during the development of higher organisms. Asymmetry is achieved by differential localization or activities of biological molecules such as proteins, and coding and non-coding RNAs. Here, we present subcellular transcriptomic and proteomic analyses along the animal-vegetal axis of Xenopus laevis eggs. More than 98% of the maternal mRNAs could be categorized into four localization profile groups: animal, vegetal, extremely vegetal, and a newly described group of mRNAs that we call extremely animal, which are mRNAs enriched in the animal cortex region. 3'UTRs of localized mRNAs were analyzed for localization motifs. Several putative motifs were discovered for vegetal and extremely vegetal mRNAs, while no distinct conserved motifs for the extremely animal mRNAs were identified, suggesting different localization mechanisms. Asymmetric profiles were also found for proteins, with correlation to those of corresponding mRNAs. Based on un...
The aim of this study was to elucidate the molecular status of single human adult germ stem cells... more The aim of this study was to elucidate the molecular status of single human adult germ stem cells (haGSCs) and haGSC colonies, which spontaneously developed from the CD49f MACS and matrix- (collagen-/laminin+ binding-) selected fraction of enriched spermatogonia. Single-cell transcriptional profiling by Fluidigm BioMark system of a long-term cultured haGSCs cluster in comparison to human embryonic stem cells (hESCs) and human fibroblasts (hFibs) revealed that haGSCs showed a characteristic germ- and pluripotency-associated gene expression profile with some similarities to hESCs and with a significant distinction from somatic hFibs. Genome-wide comparisons with microarray analysis confirmed that different haGSC colonies exhibited gene expression heterogeneity with more or less pluripotency. The results of this study confirm that haGSCs are adult stem cells with a specific molecular gene expression profile in vitro, related but not identical to true pluripotent stem cells. Under ES-ce...
MicroRNAs are a family of naturally occurring small noncoding RNA molecules that play an importan... more MicroRNAs are a family of naturally occurring small noncoding RNA molecules that play an important regulatory role in gene expression. They are suggested to regulate a large proportion of protein encoding genes by mediating the translational suppression and posttranscriptional control of gene expression. Recent findings show that microRNAs are emerging as important regulators of cellular differentiation and dedifferentiation, and are deeply involved in developmental processes including human preimplantation development. They keep a balance between pluripotency and differentiation in the embryo and embryonic stem cells. Moreover, it became evident that dysregulation of microRNA expression may play a fundamental role in progression and dissemination of different cancers including ovarian cancer. The interest is still increased by the discovery of exosomes, that is, cell-derived vesicles, which can carry different proteins but also microRNAs between different cells and are involved in ...
The advent of molecular characterization of tissues has brought an increasing emphasis on the qua... more The advent of molecular characterization of tissues has brought an increasing emphasis on the quality of biospecimens, starting with the tissue procurement process. RNA levels are particularly affected by factors in the collection process, but the influence of different pre-analytical factors is not well understood. Here we present the influence of tissue specimen size, as well as the transport and freezing protocols, on RNA quality. Large, medium, and smaller porcine liver samples were stored either dry, on moist gauze, or in salt solution for various times, and then frozen in either liquid nitrogen or in pre-cooled isopentane. Large and small human liver samples were frozen in pre-cooled isopentane either immediately or after one hour at room temperature. The small samples were stored dry, on moist gauze, or in salt solution. RNA was isolated and RIN values were measured. The RNA for six standard reference genes from human liver was analyzed by RT-qPCR, and tissue morphology was assessed for artifacts of freezing. Experiments using porcine liver samples showed that RNA derived from smaller samples was more degraded after one hour of cold ischemia, and that cooled transport is preferable. Human liver samples showed significant RNA degradation after 1 h of cold ischemia, which was more pronounced in smaller samples. RNA integrity was not significantly influenced by the transport or freezing method, but changes in gene expression were observed in samples either transported on gauze or in salt solution. Based on observations in liver samples, smaller samples are more subject to gene expression variability introduced by post-excision sample handling than are larger samples. Small biopsies should be transported on ice and snap frozen as soon as possible after acquisition from the patient.
We have examined the imprecision in the estimation of PCR efficiency by means of standard curves ... more We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1-16 qPCR replicates per concentration and we tested 2-10 l volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3-4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range.
We have measured the expression of 41 maternal mRNAs in individual blastomeres collected from the... more We have measured the expression of 41 maternal mRNAs in individual blastomeres collected from the 8 to 32-cell Xenopus laevis embryos to determine when and how asymmetry in the body plan is introduced. We demonstrate that the asymmetry along the animal-vegetal axis in the oocyte is transferred to the daughter cells during early cell divisions. All studied mRNAs are distributed evenly among the set of animal as well as vegetal blastomeres. We find no asymmetry in mRNA levels that might be ascribed to the dorso-ventral specification or the left-right axis formation. We hypothesize that while the animal-vegetal asymmetry is a consequence of mRNA gradients, the dorso-ventral and left-right axes specifications are induced by asymmetric distribution of other biomolecules, probably proteins.
The aim of this study was to trigger the expression of genes related to oocytes in putative ovari... more The aim of this study was to trigger the expression of genes related to oocytes in putative ovarian stem cells scraped from the ovarian surface epithelium of women with premature ovarian failure and cultured in vitro in the presence of follicular fluid, rich in substances for oocyte growth and maturation. Ovarian surface epithelium was scraped and cell cultures were set up by scrapings in five women with nonfunctional ovaries and with no naturally present mature follicles or oocytes. In the presence of donated follicular fluid putative stem cells grew and developed into primitive oocyte-like cells. A detailed single-cell gene expression profiling was performed to elucidate their genetic status in comparison to human embryonic stem cells, oocytes, and somatic fibroblasts. The ovarian cell cultures depleted/converted reproductive hormones from the culture medium. Estradiol alone or together with other substances may be involved in development of these primitive oocyte-like cells. The ...
BACKGROUND Determining the transcriptional profile of circulating tumor cells (CTCs) may allow th... more BACKGROUND Determining the transcriptional profile of circulating tumor cells (CTCs) may allow the acquisition of clinically relevant information while overcoming tumor heterogeneity-related biases associated with use of tissue samples for biomarker assessment. However, such molecular characterization is challenging because CTCs are rare and outnumbered by blood cells. METHODS Here, we describe a technical protocol to measure the expression of >29 000 genes in CTCs captured from whole blood with magnetic beads linked with antibodies against epithelial cell adhesion molecule (EpCAM) and the carcinoma-associated mucin, MUC1, designed to be used for CTC characterization in clinical samples. Low numbers of cells (5–200) from the MCF7 and MDA-MB-468 breast cancer cell lines were spiked in healthy donor blood samples and isolated with the AdnaTest EMT-1/Stem CellSelect kit. Gene expression profiles (GEPs) were obtained with the WG-DASL HT assay and compared with GEPs obtained from RNA ...
In healthy humans, 60–70% of the B lymphocytes produce kappa light chains, while the remaining ce... more In healthy humans, 60–70% of the B lymphocytes produce kappa light chains, while the remaining cells produce lambda light chains. Malignant transformation and clonal expansion of B lymphocytes lead to an altered kappa : lambda expression ratio, which is an important diagnostic criteria of lymphomas. Here, we compared three methods for clonality determination of suspected B cell lymphomas. Tumor biopsies from 55 patients with B cell malignancies, 5 B-lymphoid tumor cell lines, and 20 biopsies from patients with lymphadenitis were analyzed by immunohistochemistry, flow cytometry, and reverse transcription quantitative real-time PCR. Clonality was determined by immunohistochemistry in 52/53 cases, flow cytometry in 30/39 cases, and reverse transcription quantitative real-time PCR in 33/55 cases. In conclusion, immunohistochemistry was superior to flow cytometry and reverse transcription quantitative real-time PCR for clonality identification. Flow cytometry and reverse transcription qu...
There is an increasing need for proper quality control tools in the pre-analytical phase of the m... more There is an increasing need for proper quality control tools in the pre-analytical phase of the molecular diagnostic workflow. The aim of the present study was to identify biomarkers for monitoring pre-analytical mRNA quality variations in two different types of blood collection tubes, K2EDTA (EDTA) tubes and PAXgene Blood RNA Tubes (PAXgene tubes). These tubes are extensively used both in the diagnostic setting as well as for research biobank samples. Blood specimens collected in the two different blood collection tubes were stored for varying times at different temperatures, and microarray analysis was performed on resultant extracted RNA. A large set of potential mRNA quality biomarkers for monitoring post-phlebotomy gene expression changes and mRNA degradation in blood was identified. qPCR assays for the potential biomarkers and a set of relevant reference genes were generated and used to pre-validate a sub-set of the selected biomarkers. The assay precision of the potential qPC...
One purpose of the EC funded project, SPIDIA, is to develop evidence-based quality guidelines for... more One purpose of the EC funded project, SPIDIA, is to develop evidence-based quality guidelines for the pre-analytical handling of blood samples for RNA molecular testing. To this end, two pan-European External Quality Assessments (EQAs) were implemented. Here we report the results of the second SPIDIA-RNA EQA. This second study included modifications in the protocol related to the blood collection process, the shipping conditions and pre-analytical specimen handling for participants. Participating laboratories received two identical proficiency blood specimens collected in tubes with or without an RNA stabilizer. For pre-defined specimen storage times and temperatures, laboratories were asked to perform RNA extraction from whole blood according to their usual procedure and to return extracted RNA to the SPIDIA facility for further analysis. These RNA samples were evaluated for purity, yield, integrity, stability, presence of interfering substances, and gene expression levels for the ...
Real-time PCR tomography is a novel, quantitative method for measuring localized RNA expression p... more Real-time PCR tomography is a novel, quantitative method for measuring localized RNA expression profiles within single cells. We demonstrate its usefulness by dissecting an oocyte from Xenopus laevis into slices along its animal-vegetal axis, extracting its RNA and measuring the levels of 18 selected mRNAs by real-time RT-PCR. This identified two classes of mRNA, one preferentially located towards the animal, the other towards the vegetal pole. mRNAs within each group show comparable intracellular gradients, suggesting they are produced by similar mechanisms. The polarization is substantial, though not extreme, with around 5% of vegetal gene mRNA molecules detected at the animal pole, and around 50% of the molecules in the far most vegetal section. Most animal pole mRNAs were found in the second section from the animal pole and in the central section, which is where the nucleus is located. mRNA expression profiles did not change following in vitro fertilization and we conclude that the cortical rotation that follows fertilization has no detectable effect on intracellular mRNA gradients.
Uploads
Papers by Mikael Kubista