Applications like drug development need simple and streamlined methods to process samples from 96-well cell culture plates for gene expression measurements. Unfortunately, current options are expensive for such processing. Therefore, our aim was to develop a method that would allow streamlined analysis of mRNA from 96-well cell culture plates while being relatively cheap and simple. We developed a method based on the qPCR 'Cells-to-cDNA' approach and validated it against commercially available kits using the same approach or spin columns-based RNA purification. For this purpose, we conducted a series of comparisons of gene expression from peripheral blood mononuclear cells, SK-HEP-1 and U-87 cell cultures in 96-well plates. Our final method involved lysing cells with 25-100 μl solution of 0.5% SDS, 10 mM DTT, 1 mg ml-1 proteinase K dissolved in water, 1 h incubation at 50°C, followed by proteinase K inactivation at 90°C for 5 min and lysate neutralization with 1 : 1 dilution by 20% Tween 20 solution. Reverse transcription and qPCR were carried out using standard methods. This method showed a mean reduction of Ct ± s.d. value by 2.4 ± 1.3 compared with the 'Cells-to-cDNA' kit and by 1.4 ± 0.5 compared with the RNA purification kit with lower variability.
- MeSH
- Cost-Benefit Analysis MeSH
- Cell Culture Techniques methods economics MeSH
- DNA, Complementary * genetics MeSH
- Real-Time Polymerase Chain Reaction methods MeSH
- Leukocytes, Mononuclear cytology metabolism MeSH
- Humans MeSH
- RNA, Messenger genetics metabolism MeSH
- Cell Line, Tumor MeSH
- Gene Expression Profiling methods economics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The recent human Monkeypox outbreak underlined the importance of studying basic biology of orthopoxviruses. However, the transcriptome of its causative agent has not been investigated before neither with short-, nor with long-read sequencing approaches. This Oxford Nanopore long-read RNA-Sequencing dataset fills this gap. It will enable the in-depth characterization of the transcriptomic architecture of the monkeypox virus, and may even make possible to annotate novel host transcripts. Moreover, our direct cDNA and native RNA sequencing reads will allow the estimation of gene expression changes of both the virus and the host cells during the infection. Overall, our study will lead to a deeper understanding of the alterations caused by the viral infection on a transcriptome level.
- MeSH
- DNA, Complementary MeSH
- Humans MeSH
- Nanopore Sequencing * MeSH
- Mpox, Monkeypox * MeSH
- Gene Expression Profiling MeSH
- Transcriptome MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Dataset MeSH
CONTEXT: Testing for BCR-ABL1 kinase domain (KD) mutations should always be performed before tyrosine kinase inhibitor (TKI) changes. Next-generation sequencing (NGS) is the best approach to highlight emerging mutations in patients not responding adequately to TKI therapy. However, NGS requires sample centralization and batch analysis and has a non-negligible time to results. In this study, we set up and validated a novel droplet digital PCR (ddPCR)-based multiplex strategy for the detection and quantitation of transcripts harboring mutations impacting TKI selection. METHODS: In collaboration with Bio-Rad, a 3-tube ddPCR strategy was designed that enables identification and quantitation of 16 nucleotide substitutions encoding the 13 mutations associated with resistance to one or more second-generation TKI (2GTKI). Primers and FAM-or FAM/HEX-labelled probes were grouped on a TKI-specific basis and generated clusters of droplets mapping to spatially distinct areas of the 2D plot based on resistance profiles. Each tube also incorporated primers and HEX-labelled probes for e13a2, e14a2, and e1a2 BCR::ABL1 fusion transcripts to express results as the percentage of mutation-positive over total BCR::ABL1 transcripts. For validation, a total of 101 RNA samples from healthy donors, TKI-sensitive and -resistant patients, and BCR::ABL1-positive and -negative cell lines were used. cDNA (125 ng) obtained with ABL1-specific primers was analyzed in duplicate on a QX200 ddPCR system (Bio-Rad). RESULTS: The limit of blank was determined using 60 blank samples. Accuracy and specificity were confirmed using 48 samples positive for one or more 2GTKI-resistant mutations or mutations at nearby codons (to exclude cross-reactivity). Analysis of serial dilutions of cell line mixtures made using BCR::ABL1-positive mutation-positive cells, BCR::ABL1-positive unmutated cells, and BCR::ABL1-negative cells to mimic different mutation frequencies (70%, 5%, and 0.5%) and different transcript levels (MR0 to MR3) showed that a 0.5% lower detection limit could be consistently achieved irrespective of BCR::ABL1 levels. CONCLUSIONS: ddPCR proved highly sensitive and accurate. Total hands-on time was approximately 2 hrs, and time from sample to results was 2 days. Therefore, ddPCR may be integrated into diagnostic algorithms of CML (and Ph+ ALL) patients as a convenient first-level screening tool for mutations impacting TKI selection.
- MeSH
- Fusion Proteins, bcr-abl * genetics metabolism MeSH
- Drug Resistance, Neoplasm genetics MeSH
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive * diagnosis drug therapy genetics MeSH
- Protein Kinase Inhibitors pharmacology therapeutic use MeSH
- DNA, Complementary pharmacology MeSH
- Humans MeSH
- Mutation MeSH
- Nucleotides MeSH
- Polymerase Chain Reaction MeSH
- RNA MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Isolation of RNA from whole saliva, a non-invasive and easily accessible biofluid that is an attractive alternative to blood for high-throughput biodosimetry of radiological/nuclear victims might be of clinical significance for prediction and diagnosis of disease. In a previous analysis of 12 human samples we identified two challenges to measuring gene expression from total RNA: (1) the fraction of human RNA in whole saliva was low and (2) the bacterial contamination was overwhelming. To overcome these challenges, we performed selective cDNA synthesis for human RNA species only by employing poly(A)+-tail primers followed by qRT-PCR. In the current study, this approach was independently validated on 91 samples from 61 healthy donors. Additionally, we used the ratio of human to bacterial RNA to adjust the input RNA to include equal amounts of human RNA across all samples before cDNA synthesis, which then ensured comparable analysis using the same base human input material. Furthermore, we examined relative levels of ten known housekeeping genes, and assessed inter- and intra-individual differences in 61 salivary RNA isolates, while considering effects of demographical factors (e.g. sex, age), epidemiological factors comprising social habits (e.g. alcohol, cigarette consumption), oral hygiene (e.g. flossing, mouthwash), previous radiological diagnostic procedures (e.g. number of CT-scans) and saliva collection time (circadian periodic). Total human RNA amounts appeared significantly associated with age only (P ≤ 0.02). None of the chosen housekeeping genes showed significant circadian periodicity and either did not associate or were weakly associated with the 24 confounders examined, with one exception, 60% of genes were altered by mouthwash. ATP6, ACTB and B2M represented genes with the highest mean baseline expression (Ct-values ≤ 30) and were detected in all samples. Combining these housekeeping genes for normalization purposes did not decrease inter-individual variance, but increased the robustness. In summary, our work addresses critical confounders and provides important information for the successful examination of gene expression in human whole saliva.
- MeSH
- RNA, Bacterial MeSH
- Adult MeSH
- Genes, Essential * MeSH
- Gene Expression * MeSH
- DNA, Complementary MeSH
- DNA Contamination MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- RNA isolation & purification MeSH
- Saliva metabolism MeSH
- Gene Expression Profiling methods MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Element content and expression of genes of interest on single cell types, such as stomata, provide valuable insights into their specific physiology, improving our understanding of leaf gas exchange regulation. We investigated how far differences in stomatal conductance (gs ) can be ascribed to changes in guard cells functioning in amphistomateous leaves. gs was measured during the day on both leaf sides, on well-watered and drought-stressed trees (two Populus euramericana Moench and two Populus nigra L. genotypes). In parallel, guard cells were dissected for element content and gene expressions analyses. Both were strongly arranged according to genotype, and drought had the lowest impact overall. Normalizing the data by genotype highlighted a structure on the basis of leaf sides and time of day both for element content and gene expression. Guard cells magnesium, phosphorus, and chlorine were the most abundant on the abaxial side in the morning, where gs was at the highest. In contrast, genes encoding H+ -ATPase and aquaporins were usually more abundant in the afternoon, whereas genes encoding Ca2+ -vacuolar antiporters, K+ channels, and ABA-related genes were in general more abundant on the adaxial side. Our work highlights the unique physiology of each leaf side and their analogous rhythmicity through the day.
- MeSH
- Genotype MeSH
- DNA, Complementary genetics isolation & purification MeSH
- Plant Leaves genetics metabolism MeSH
- Electron Probe Microanalysis MeSH
- Droughts MeSH
- Populus classification genetics metabolism MeSH
- Proton-Translocating ATPases genetics metabolism MeSH
- Plant Stomata genetics metabolism MeSH
- Gene Expression Regulation, Plant MeSH
- RNA, Plant genetics isolation & purification MeSH
- Plant Proteins genetics metabolism MeSH
- Trees genetics metabolism MeSH
- Plant Transpiration physiology MeSH
- Water physiology MeSH
- Plant Development MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Saliva, as a non-invasive and easily accessible biofluid, has been shown to contain RNA biomarkers for prediction and diagnosis of several diseases. However, systematic analysis done by our group identified two problematic issues not coherently described before: (1) most of the isolated RNA originates from the oral microbiome and (2) the amount of isolated human RNA is comparatively low. The degree of bacterial contamination showed ratios up to 1:900,000, so that only about one out of 900,000 RNA copies was of human origin, but the RNA quality (average RIN 6.7 + /- 0.8) allowed for qRT-PCR. Using 12 saliva samples from healthy donors, we modified the methodology to (1) select only human RNA during cDNA synthesis by aiming at the poly(A)+-tail and (2) introduced a pre-amplification of human RNA before qRT-PCR. Further, the manufacturer's criteria for successful pre-amplification (Ct values ≤ 35 for unamplified cDNA) had to be replaced by (3) proofing linear pre-amplification for each gene, thus, increasing the number of evaluable samples up to 70.6%. When considering theses three modifications unbiased gene expression analysis on human salivary RNA can be performed.
- MeSH
- RNA, Bacterial genetics MeSH
- Adult MeSH
- Gene Expression * MeSH
- DNA, Complementary genetics MeSH
- Real-Time Polymerase Chain Reaction methods MeSH
- Humans MeSH
- RNA, Ribosomal, 18S genetics MeSH
- RNA analysis MeSH
- Saliva chemistry metabolism MeSH
- Gene Expression Profiling methods MeSH
- Transcriptome MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Haemophilus Influenza leads to respiratory infections such as sinusitis, acute otitis media, pneumonia and bronchitis. In addition, it causes invasive infections such as cellulite, septic arthritis, and meningitis. Therefore, quick and sensitive detection of H. influenza is of great importance in medical microbiology. In this study, a novel DNA-based bioassay was developed to the monitoring of Haemophilus influenza genome in human plasma samples using binding of pDNA with cDNA. DNA hybridization strategy was used to investigation of DNAs binding. For this purpose, silver nanoparticle doped graphene quantum dots inks functionalized by D-penicillamine (Ag NPs-DPA-GQDs) were synthesized and deposited on the surface of glass carbon electrode (GCE). Also, gold nanoparticles functionalized with cysteamine (CysA-AuNPs) were deposited on the surface of the Ag-DPA-GQDs modified GCE. Afterward, thiolated DNA probe was immobilized on the surface of the modified electrode. DNA hybridization was monitored using square wave voltammetry (SWV) technique. Engineered genosensor indicated good performance with high specificity and sensitivity for detection of Haemophilus influenza genome. Under optimal conditions, linear range and low limit of quantitation (LLOQ) were obtained as target concentrations ranging from 1 pM-1 ZM and 1 ZM, respectively. The designed biosensor also showed high capability of discriminating one-base, two-base and three-base mismatched sequences. Also, the prepared genosensor could be easily regenerated and reused to evaluate hybridization process.
- MeSH
- Biosensing Techniques MeSH
- Electrochemical Techniques MeSH
- Genome, Bacterial * MeSH
- Haemophilus influenzae genetics MeSH
- Nucleic Acid Hybridization methods MeSH
- DNA, Complementary * MeSH
- Metal Nanoparticles ultrastructure MeSH
- Quantum Dots MeSH
- Humans MeSH
- Reproducibility of Results MeSH
- Cell-Free Nucleic Acids * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The single-celled parasite Giardia intestinalis (Diplomonadida) has two equally sized nuclei in one cell. The nuclei have been considered identical. We have previously shown that they contain different chromosomal sets and proceed through the cell cycle with some asynchrony. Here, we demonstrate by fluorescence in situ hybridization that several genes from chromosome 5 are lost in one of the two nuclei of the WBc6 Giardia line. The missing segment stretches over at least 50kb near the 5' chromosome end. In both WB and WBc6 Giardia cell lines, chromosome 5 is trisomic in one nucleus and monosomic in the other nucleus. The described chromosomal deletion has always been observed at the monosomic chromosome in WBc6; however, the deletion was not detected in the parent line WB. The chromosomal segment was thus initially lost after biological cloning of WB, which gave rise to clone WBc6. We show that Giardia is capable of carrying out gene expression from only one nucleus. The two nuclei display a certain level of diversity, making each of them irreplaceable. The doubled karyomastigonts of diplomonads likely have separate functions both in the mastigont/flagellar organization and in chromosomal and gene content. To our knowledge, our results offer the first methodical approach to differentiating the two, so far indistinguishable nuclei.
- MeSH
- Cell Nucleus genetics MeSH
- Time Factors MeSH
- Chromosome Deletion MeSH
- Gene Deletion MeSH
- Giardia lamblia * genetics ultrastructure MeSH
- In Situ Hybridization, Fluorescence standards MeSH
- DNA, Complementary MeSH
- Real-Time Polymerase Chain Reaction methods MeSH
- Mitosis MeSH
- Monosomy * MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Gene Expression Regulation physiology MeSH
- Reverse Transcription MeSH
- RNA, Protozoan genetics isolation & purification MeSH
- Signal Transduction MeSH
- Trisomy genetics MeSH
The single-celled parasite Giardia intestinalis (Diplomonadida) has two equally sized nuclei in one cell. The nuclei have been considered identical. We have previously shown that they contain different chromosomal sets and proceed through the cell cycle with some asynchrony. Here, we demonstrate by fluorescence in situ hybridization that several genes from chromosome 5 are lost in one of the two nuclei of the WBc6 Giardia line. The missing segment stretches over at least 50 kb near the 5' chromosome end. In both WB and WBc6 Giardia cell lines, chromosome 5 is trisomic in one nucleus and monosomic in the other nucleus. The described chromosomal deletion has always been observed at the monosomic chromosome in WBc6; however, the deletion was not detected in the parent line WB. The chromosomal segment was thus initially lost after biological cloning of WB, which gave rise to clone WBc6. We show that Giardia is capable of carrying out gene expression from only one nucleus. The two nuclei display a certain level of diversity, making each of them irreplaceable. The doubled karyomastigonts of diplomonads likely have separate functions both in the mastigont/flagellar organization and in chromosomal and gene content. To our knowledge, our results offer the first methodical approach to differentiating the two, so far indistinguishable nuclei.
- MeSH
- Cell Nucleus genetics MeSH
- Time Factors MeSH
- Chromosome Deletion MeSH
- Gene Deletion MeSH
- Giardia lamblia genetics ultrastructure MeSH
- In Situ Hybridization, Fluorescence standards MeSH
- DNA, Complementary genetics MeSH
- Real-Time Polymerase Chain Reaction methods MeSH
- Mitosis MeSH
- Monosomy * genetics MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Gene Expression Regulation physiology MeSH
- Reverse Transcription MeSH
- RNA, Protozoan genetics isolation & purification MeSH
- Signal Transduction MeSH
- Trisomy * genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
KEY MESSAGE: The knowledge of substrate specificity of XET enzymes is important for the general understanding of metabolic pathways to challenge the established notion that these enzymes operate uniquely on cellulose-xyloglucan networks. Xyloglucan xyloglucosyl transferases (XETs) (EC 2.4.1.207) play a central role in loosening and re-arranging the cellulose-xyloglucan network, which is assumed to be the primary load-bearing structural component of plant cell walls. The sequence of mature TmXET6.3 from Tropaeolum majus (280 residues) was deduced by the nucleotide sequence analysis of complete cDNA by Rapid Amplification of cDNA Ends, based on tryptic and chymotryptic peptide sequences. Partly purified TmXET6.3, expressed in Pichia occurred in N-glycosylated and unglycosylated forms. The quantification of hetero-transglycosylation activities of TmXET6.3 revealed that (1,3;1,4)-, (1,6)- and (1,4)-β-D-glucooligosaccharides were the preferred acceptor substrates, while (1,4)-β-D-xylooligosaccharides, and arabinoxylo- and glucomanno-oligosaccharides were less preferred. The 3D model of TmXET6.3, and bioinformatics analyses of identified and putative plant xyloglucan endotransglycosylases (XETs)/hydrolases (XEHs) of the GH16 family revealed that H94, A104, Q108, K234 and K237 were the key residues that underpinned the acceptor substrate specificity of TmXET6.3. Compared to the wild-type enzyme, the single Q108R and K237T, and double-K234T/K237T and triple-H94Q/A104D/Q108R variants exhibited enhanced hetero-transglycosylation activities with xyloglucan and (1,4)-β-D-glucooligosaccharides, while those with (1,3;1,4)- and (1,6)-β-D-glucooligosaccharides were suppressed; the incorporation of xyloglucan to (1,4)-β-D-glucooligosaccharides by the H94Q variant was influenced most extensively. Structural and biochemical data of non-specific TmXET6.3 presented here extend the classic XET reaction mechanism by which these enzymes operate in plant cell walls. The evaluations of TmXET6.3 transglycosylation activities and the incidence of investigated residues in other members of the GH16 family suggest that a broad acceptor substrate specificity in plant XET enzymes could be more widespread than previously anticipated.
- MeSH
- Phylogeny MeSH
- Glycosylation MeSH
- Glycosyltransferases chemistry metabolism MeSH
- Germination MeSH
- DNA, Complementary genetics MeSH
- Models, Molecular MeSH
- Petroselinum enzymology MeSH
- Protein Engineering * MeSH
- Plant Proteins chemistry metabolism MeSH
- Amino Acid Sequence MeSH
- Base Sequence MeSH
- Seeds enzymology MeSH
- Structural Homology, Protein MeSH
- Substrate Specificity MeSH
- Tropaeolum enzymology MeSH
- Publication type
- Journal Article MeSH
