Browsing by Subject "NORMALIZATION"

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  • Mietola, Reetta; Vehmas, Simo (2019)
    This paper discusses youth and the significance of age in the lives of persons with profound intellectual and multiple disabilities. The analysis is based on an ethnographic research project that explores what makes a good life for this group of people. The findings indicate that whilst the meaning and significance of youth and age were discussed often by care workers and family members, age had very little significance in the lives of our research participants. Youth as a phase of life gets lost in the transition from children's services to adult services: age in the lives of persons with profound intellectual disabilities means merely a move from one service system to another. For the care workers, age provides a way to evaluate and criticize the service system and whether it caters for the individual needs of persons with profound intellectual disabilities.
  • Tang, Zhipeng; Adhikari, Hari; Pellikka, Petri; Heiskanen, Janne (2021)
    Combined with increasing computing ability, the free and open access to Landsat archive has enabled the changes on the Earth’s surface to be monitored for almost 50 years. However, due to missing observations that result from clouds, cloud shadows, and scan line corrector failure, the Landsat data record is neither a continuous nor consistent time series. We present a new gap-filling method, Missing Observation Prediction based on Spectral-Temporal Metrics (MOPSTM), which uses spectral-temporal metrics computed from Landsat one-year time series and the k-Nearest Neighbor (k-NN) regression. Herein, we demonstrate the performance of MOPSTM by using five, nearly cloud-free, full scene Landsat images from Kenya, Finland, Germany, the USA, and China. Cloud masks from the images with extensive cloud cover were used to simulate large-area gaps, with the highest value we tested being 92% of missing data. The gap-filling accuracy was assessed quantitatively considering all five sites and different land use/land cover types, and the MOPSTM algorithm performed better than the spectral angle-mapper based spatiotemporal similarity (SAMSTS) gap-filling algorithm. The mean RMSE values of MOPSTM were 0.010, 0.012, 0.025, 0.012, and 0.018 for the five sites, while those of SAMSTS were 0.011, 0.017, 0.038, 0.014, and 0.023, respectively. Furthermore, MOPSTM had mean coefficient of determination (R2) values of 0.90, 0.86, 0.78, 0.92, and 0.89, which were higher than those for SAMSTS (0.84, 0.75, 0.55, 0.89, and 0.83). The performance of MOPSTM was not considerably affected by image gap sizes as images ranging from gap sizes of 51% of the image all the way to 92% of the image yielded similar gap-filling accuracy. Also, MOPSTM does not require local parametertuning except for the k values in the k-NN regression, and it can make a gap-free image from any acquisition date. MOPSTM provides a new spectral-temporal approach to generate the gap-free imagery for typical Landsat applications, such as land use, land cover, and forest monitoring.
  • Li, Zhilin; Korhonen, Emilia A.; Merlini, Arianna; Strauss, Judith; Wihuri, Eleonoora; Nurmi, Harri; Antila, Salli; Paech, Jennifer; Deutsch, Urban; Engelhardt, Britta; Chintharlapalli, Sudhakar; Koh, Gou Young; Flügel, Alexander; Alitalo, Kari (2020)
    Angiopoietin-2 (Ang2), a ligand of the endothelial Tie2 tyrosine kinase, is involved in vascular inflammation and leakage in critically ill patients. However, the role of Ang2 in demyelinating central nervous system (CNS) autoimmune diseases is unknown. Here, we report that Ang2 is critically involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis. Ang2 expression was induced in CNS autoimmunity, and transgenic mice overexpressing Ang2 specifically in endothelial cells (ECs) developed a significantly more severe EAE. In contrast, treatment with Ang2-blocking Abs ameliorated neuroinflammation and decreased spinal cord demyelination and leukocyte infiltration into the CNS. Similarly, Ang2-binding and Tie2-activating Ab attenuated the development of CNS autoimmune disease. Ang2 blockade inhibited expression of EC adhesion molecules, improved blood-brain barrier integrity, and decreased expression of genes involved in antigen presentation and proinflammatory responses of microglia and macrophages, which was accompanied by inhibition of α5β1 integrin activation in microglia. Taken together, our data suggest that Ang2 provides a target for increasing Tie2 activation in ECs and inhibiting proinflammatory polarization of CNS myeloid cells via α5β1 integrin in neuroinflammation. Thus, Ang2 targeting may serve as a therapeutic option for the treatment of CNS autoimmune disease.
  • Gupta, Rashi; Greco, Dario; Auvinen, Petri; Arjas, Elja (2010)
  • Chmielarz, Piotr; Konovalova, Julia; Najam, Syeda Sadia; Alter, Heike; Piepponen, Timo Petteri; Erfle, Holger; Sonntag, Kai C.; Schutz, Gunther; Vinnikov, Ilya A.; Domanskyi, Andrii (2017)
    MicroRNAs (miRs) are important post-transcriptional regulators of gene expression implicated in neuronal development, differentiation, aging and neurodegenerative diseases, including Parkinson's disease (PD). Several miRs have been linked to PD-associated genes, apoptosis and stress response pathways, suggesting that deregulation of miRs may contribute to the development of the neurodegenerative phenotype. Here, we investigate the cell-autonomous role of miR processing RNAse Dicer in the functional maintenance of adult dopamine (DA) neurons. We demonstrate a reduction of Dicer in the ventral midbrain and altered miR expression profiles in laser-microdissected DA neurons of aged mice. Using a mouse line expressing tamoxifen-inducible CreERT2 recombinase under control of the DA transporter promoter, we show that a tissue-specific conditional ablation of Dicer in DA neurons of adult mice led to decreased levels of striatal DA and its metabolites without a reduction in neuronal body numbers in hemizygous mice (Dicer(HET)) and to progressive loss of DA neurons with severe locomotor deficits in nullizygous mice (Dicer(CKO)). Moreover, we show that pharmacological stimulation of miR biosynthesis promoted survival of cultured DA neurons and reduced their vulnerability to thapsigargin-induced endoplasmic reticulum stress. Our data demonstrate that Dicer is crucial for maintenance of adult DA neurons, whereas a stimulation of miR production can promote neuronal survival, which may have direct implications for PD treatment.
  • Fleischer, Thomas; Klajic, Jovana; Aure, Miriam Ragle; Louhimo, Riku; Pladsen, Arne V.; Ottestad, Lars; Touleimat, Nizar; Laakso, Marko; Halvorsen, Ann Rita; Alnaes, Grethe I. Grenaker; Riis, Margit L. H.; Helland, Aslaug; Hautaniemi, Sampsa; Lonning, Per Eystein; Naume, Bjorn; Borresen-Dale, Anne-Lise; Tost, Joerg; Kristensen, Vessela N. (2017)
    Breast cancer patients with Luminal A disease generally have a good prognosis, but among this patient group are patients with good prognosis that are currently overtreated with adjuvant chemotherapy, and also patients that have a bad prognosis and should be given more aggressive treatment. There is no available method for subclassification of this patient group. Here we present a DNA methylation signature (SAM40) that segregates Luminal A patients based on prognosis, and identify one good prognosis group and one bad prognosis group. The prognostic impact of SAM40 was validated in four independent patient cohorts. Being able to subdivide the Luminal A patients may give the two-sided benefit of identifying one subgroup that may benefit from a more aggressive treatment than what is given today, and importantly, identifying a subgroup that may benefit from less treatment.
  • Korpela, Ilkka; Haapanen, R.; Korrensalo, A.; Tuittila, E-S; Vesala, T. (2020)
    Boreal bogs are important stores and sinks of atmospheric carbon whose surfaces are characterised by vegetation microforms. Efficient methods for monitoring their vegetation are needed because changes in vegetation composition lead to alteration in their function such as carbon gas exchange with the atmosphere. We investigated how airborne image and waveform-recording LiDAR data can be used for 3D mapping of microforms in an open bog which is a mosaic of pools, hummocks with a few stunted pines, hollows, intermediate surfaces and mud-bottom hollows. The proposed method operates on the bog surface, which is reconstructed using LiDAR. The vegetation was classified at 20 cm resolution. We hypothesised that LiDAR data describe surface topography, moisture and the presence and depth of field-layer vegetation and surface roughness; while multiple images capture the colours and texture of the vegetation, which are influenced by directional reflectance effects. We conclude that geometric LiDAR features are efficient predictors of microforms. LiDAR intensity and echo width were specific to moisture and surface roughness, respectively. Directional reflectance constituted 4-34 % of the variance in images and its form was linked to the presence of the field layer. Microform-specific directional reflectance patterns were deemed to be of marginal value in enhancing the classification, and RGB image features were inferior to LiDAR variables. Sensor fusion is an attractive option for fine-scale mapping of these habitats. We discuss the task and propose options for improving the methodology.
  • Korber, Inken; Katayama, Shintaro; Einarsdottir, Elisabet; Krjutskov, Kaarel; Hakala, Paula; Kere, Juha; Lehesjoki, Anna-Elina; Joensuu, Tarja (2016)
    Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1, OMIM254800) is an autosomal recessive neurodegenerative disorder characterized by stimulus-sensitive and action-activated myoclonus, tonic-clonic epileptic seizures, and ataxia. Loss-of-function mutations in the gene encoding the cysteine protease inhibitor cystatin B (CSTB) underlie EPM1. The deficiency of CSTB in mice (Cstb(-/-) mice) generates a phenotype resembling the symptoms of EPM1 patients and is accompanied by microglial activation at two weeks of age and an upregulation of immune system-associated genes in the cerebellum at one month of age. To shed light on molecular pathways and processes linked to CSTB deficiency in microglia we characterized the transcriptome of cultured Cstb(-/-) mouse microglia using microarray hybridization and RNA sequencing (RNA-seq). The gene expression profiles obtained with these two techniques were in good accordance and not polarized to either pro- or anti-inflammatory status. In Cstb(-/-) microglia, altogether 184 genes were differentially expressed. Of these, 33 genes were identified by both methods. Several interferon-regulated genes were weaker expressed in Cstb(-/-) microglia compared to control. This was confirmed by quantitative real-time PCR of the transcripts Irf7 and Stat1. Subsequently, we explored the biological context of CSTB deficiency in microglia more deeply by functional enrichment and canonical pathway analysis. This uncovered a potential role for CSTB in chemotaxis, antigen-presentation, and in immune-and defense response-associated processes by altering JAK-STAT pathway signaling. These data support and expand the previously suggested involvement of inflammatory processes to the disease pathogenesis of EPM1 and connect CSTB deficiency in microglia to altered expression of interferon-regulated genes.
  • Genetics of DNA Methylation Consortium; BIOS Consortium; van Dongen, Jenny; Lundgren, Sara; Ollikainen, Miina; Kaprio, Jaakko (2021)
    The mechanisms underlying how monozygotic (or identical) twins arise are yet to be determined. Here, the authors investigate this in an epigenome-wide association study, showing that monozygotic twinning has a characteristic DNA methylation signature in adult somatic tissues. Monozygotic (MZ) twins and higher-order multiples arise when a zygote splits during pre-implantation stages of development. The mechanisms underpinning this event have remained a mystery. Because MZ twinning rarely runs in families, the leading hypothesis is that it occurs at random. Here, we show that MZ twinning is strongly associated with a stable DNA methylation signature in adult somatic tissues. This signature spans regions near telomeres and centromeres, Polycomb-repressed regions and heterochromatin, genes involved in cell-adhesion, WNT signaling, cell fate, and putative human metastable epialleles. Our study also demonstrates a never-anticipated corollary: because identical twins keep a lifelong molecular signature, we can retrospectively diagnose if a person was conceived as monozygotic twin.
  • Laurila, Kirsti; Autio, Reija; Kong, Lingjia; Narva, Elisa; Hussein, Samer; Otonkoski, Timo; Lahesmaa, Riitta; Lahdesmaki, Harri (2014)
  • Tervaniemi, Mari H.; Katayama, Shintaro; Skoog, Tiina; Siitonen, H. Annika; Vuola, Jyrki; Nuutila, Kristo; Tammimies, Kristiina; Suomela, Sari; Kankuri, Esko; Kere, Juha; Elomaa, Outi (2018)
    Background: CCHCR1 (Coiled Coil alpha-Helical Rod protein 1) is a putative psoriasis candidate gene with the risk alleles CCHCR1*WWCC and *Iso3, the latter inhibiting the translation of isoform 1. CCHCR1 was recently shown to be a centiosomal piotein, as well as a component of cytoplasmic piocessmg bodies (P-bodies) that regulate mRNA turnovel. The function of CCHCR1 has remained unsettled, partly because of the inconsistent findings, it has been shown to play a wide variety of roles in divergent processes, e.g., cell proliferation and steroidogenesis. Here we utilized RNA sequencing (RNAseq) using HEK293 cells overexpressing isoforms 1 or 3 (Iso1, Iso3 cells), in combination with the coding non-risk or risk (*WWCC haplotype of CCHCR1. Our aim was to study the overall role of CCHCR1 and the effects of its variants. Results: The overexpression of CCHCR1 variants in HEK293 cells resulted in cell line-specific expression profiles though seveial similarities were observable. Overall the Iso1 and Iso3 cells showed a clear isoform-specific clustering as two separate groups, and the Non-risk and Risk cells often exhibited opposite effects. The RNAseq supported a role for CCHCR1 in the centrosomes and P-bodies; the most highlighted pathways included regulation of cytoskeleton, adherens and tight junctions, mRNA surveillance and RNA transport. Interestingly, both the RNAseq and immunofluorescent localization revealed variant-specific differences for CCHCR1 within the P-bodies. Conclusions: CCHCR1 influenced a wide variety of signaling pathways, which could reflect its active role in the P-bodies and centrosomes that both are linked to the cytoskeleton; as a centrosomal P-body protein CCHCR1 may regulate diverse cytoskeleton-mediated functions, such as cell adhesion and division. The piesent findings may explain the previous inconsistent obseivations about the functions of CCHCR1.
  • Siironen, Päivi; Hagstrom, Jaana; Maenpaa, Hanna O.; Louhimo, Johanna; Arola, Johanna; Haglund, Caj (2016)
    Background Total thyroidectomy is the treatment of choice for medullary thyroid carcinoma (MTC), but the extent of neck dissection is controversial. Lymph node metastases, distant metastases, and old age are known predictors of poor survival. Patients Patients treated for primary MTC at Helsinki University Hospital from 1990 to 2009 were included (n = 54). Their clinical characteristics, treatment, and outcome were analysed retrospectively, these patients were followed until death or their last follow-up date. Results At last follow-up (3.4-23 years), of 54 MTC patients, 19 (35%) were disease-free, 17 (32%) were alive with disease, and 12 (22%) had died of MTC; six patients died of unrelated causes (11%). All disease-free patients were node negative and had normal postoperative calcitonin level. Of 19 disease-free patients, only four (21%) had undergone lymph node dissection. All patients who died of MTC were Stage IV at diagnosis and died with distant metastases. Disease-specific five-and 10-year survival was 84% and 76.2%. Advanced T-stage (p = 0.004), lymph node metastases (p <0.001), distant metastases (p <0.001), stage (p <0.001), and elevated postoperative calcitonin (p <0.001) significantly associated with survival. Conclusions Lymph node metastasis and elevated postoperative calcitonin are important prognostic factors. Patients with lymph node metastasis and/or elevated postoperative calcitonin with present treatments cannot become disease-free, but most of them can live a long life with metastasis.
  • Hakonen, Elina; Chandra, Vikash; Fogarty, Christopher L.; Yu, Nancy Yiu-Lin; Ustinov, Jarkko; Katayama, Shintaro; Galli, Emilia; Danilova, Tatiana; Lindholm, Paivi; Vartiainen, Aki; Einarsdottir, Elisabet; Krjutskov, Kaarel; Kere, Juha; Saarma, Mart; Lindahl, Maria; Otonkoski, Timo (2018)
    There is a great need to identify factors that could protect pancreatic beta cells against apoptosis or stimulate their replication and thus prevent or reverse the development of diabetes. One potential candidate is mesencephalic astrocyte-derived neurotrophic factor (MANF), an endoplasmic reticulum (ER) stress inducible protein. Manf knockout mice used as a model of diabetes develop the condition because of increased apoptosis and reduced proliferation of beta cells, apparently related to ER stress. Given this novel association between MANF and beta cell death, we studied the potential of MANF to protect human beta cells against experimentally induced ER stress. Primary human islets were challenged with proinflammatory cytokines, with or without MANF. Cell viability was analysed and global transcriptomic analysis performed. Results were further validated using the human beta cell line EndoC-beta H1. There was increased expression and secretion of MANF in human beta cells in response to cytokines. Addition of recombinant human MANF reduced cytokine-induced cell death by 38% in human islets (p <0.05). MANF knockdown in EndoC-beta H1 cells led to increased ER stress after cytokine challenge. Mechanistic studies showed that the protective effect of MANF was associated with repression of the NF-kappa B signalling pathway and amelioration of ER stress. MANF also increased the proliferation of primary human beta cells twofold when TGF-beta signalling was inhibited (p <0.01). Our studies show that exogenous MANF protein can provide protection to human beta cells against death induced by inflammatory stress. The antiapoptotic and mitogenic properties of MANF make it a potential therapeutic agent for beta cell protection.
  • Aho, Hanna; Pietila, Ilkka; Joronen, Katja (2019)
  • Krjutskov, K.; Katayama, S.; Saare, M.; Vera-Rodriguez, M.; Lubenets, D.; Samuel, K.; Laisk-Podar, T.; Teder, H.; Einarsdottir, E.; Salumets, A.; Kere, J. (2016)
    STUDY QUESTION: How can we study the full transcriptome of endometrial stromal and epithelial cells at the single-cell level? SUMMARY ANSWER: By compiling and developing novel analytical tools for biopsy, tissue cryopreservation and disaggregation, single-cell sorting, library preparation, RNA sequencing (RNA-seq) and statistical data analysis. WHAT IS KNOWN ALREADY: Although single-cell transcriptome analyses from various biopsied tissues have been published recently, corresponding protocols for human endometrium have not been described. STUDY DESIGN, SIZE, DURATION: The frozen-thawed endometrial biopsies were fluorescence-activated cell sorted (FACS) to distinguish CD13-positive stromal and CD9-positive epithelial cells and single-cell transcriptome analysis performed from biopsied tissues without culturing the cells. We studied gene transcription, applying a modern and efficient RNA-seq protocol. In parallel, endometrial stromal cells were cultured and global expression profiles were compared with uncultured cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: For method validation, we used two endometrial biopsies, one from mid-secretory phase (Day 21, LH+8) and another from late-secretory phase (Day 25). The samples underwent single-cell FACS sorting, single-cell RNA-seq library preparation and Illumina sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: Here we present a complete pipeline for single-cell gene-expression studies, from clinical sampling to statistical data analysis. Tissue manipulation, starting from disaggregation and cell-type-specific labelling and ending with single-cell automated sorting, is managed within 90 min at low temperature to minimize changes in the gene expression profile. The single living stromal and epithelial cells were sorted using CD13- and CD9-specific antibodies, respectively. Of the 8622 detected genes, 2661 were more active in cultured stromal cells than in biopsy cells. In the comparison of biopsy versus cultured cells, 5603 commonly expressed genes were detected, with 241 significantly differentially expressed genes. Of these, 231 genes were up- and 10 down-regulated in cultured cells, respectively. In addition, we performed a gene ontology analysis of the differentially expressed genes and found that these genes are mainly related to cell cycle, translational processes and metabolism. LIMITATIONS, REASONS FOR CAUTION: Although CD9-positive single epithelial cells sorting was successfully established in our laboratory, the amount of transcriptome data per individual epithelial cell was low, complicating further analysis. This step most likely failed due to the high dose of RNases that are released by the cells' natural processes, or due to rapid turnaround time or the apoptotic conditions in freezing- or single-cell solutions. Since only the cells from the late-secretory phase were subject to more focused analysis, further studies including larger sample size from the different time-points of the natural menstrual cycle are needed. The methodology also needs further optimization to examine different cell types at high quality. WIDER IMPLICATIONS OF THE FINDINGS: The symbiosis between clinical biopsy and the sophisticated laboratory and bioinformatic protocols described here brings together clinical diagnostic needs and modern laboratory and bioinformatic solutions, enabling us to implement a precise analytical toolbox for studying the endometrial tissue even at the single-cell level.
  • Yu, Xiaowei; Hyyppa, Juha; Litkey, Paula; Kaartinen, Harri; Vastaranta, Mikko; Holopainen, Markus (2017)
    This paper investigated the potential of multispectral airborne laser scanning (ALS) data for individual tree detection and tree species classification. The aim was to develop a single-sensor solution for forest mapping that is capable of providing species-specific information, required for forest management and planning purposes. Experiments were conducted using 1903 ground measured trees from 22 sample plots and multispectral ALS data, acquired with an Optech Titan scanner over a boreal forest, mainly consisting of Scots pine (Pinus Sylvestris), Norway spruce (Picea Abies), and birch (Betula sp.), in southern Finland. ALS-features used as predictors for tree species were extracted from segmented tree objects and used in random forest classification. Different combinations of features, including point cloud features, and intensity features of single and multiple channels, were tested. Among the field-measured trees, 61.3% were correctly detected. The best overall accuracy (OA) of tree species classification achieved for correctly-detected trees was 85.9% (Kappa = 0.75), using a point cloud and single-channel intensity features combination, which was not significantly different from the ones that were obtained either using all features (OA = 85.6%, Kappa = 0.75), or single-channel intensity features alone (OA = 85.4%, Kappa = 0.75). Point cloud features alone achieved the lowest accuracy, with an OA of 76.0%. Field-measured trees were also divided into four categories. An examination of the classification accuracy for four categories of trees showed that isolated and dominant trees can be detected with a detection rate of 91.9%, and classified with a high overall accuracy of 90.5%. The corresponding detection rate and accuracy were 81.5% and 89.8% for a group of trees, 26.4% and 79.1% for trees next to a larger tree, and 7.2% and 53.9% for trees situated under a larger tree, respectively. The results suggest that Channel 2 (1064 nm) contains more information for separating pine, spruce, and birch, followed by channel 1 (1550 nm) and channel 3 (532 nm) with an overall accuracy of 81.9%, 78.3%, and 69.1%, respectively. Our results indicate that the use of multispectral ALS data has great potential to lead to a single-sensor solution for forest mapping.
  • Linares, Nancy Coconi; Di Falco, Marcos; Benoit-Gelber, Isabelle; Gruben, Birgit S.; Peng, Mao; Tsang, Adrian; Mäkelä, Miia R.; de Vries, Ronald P. (2019)
    Guar gum consists mainly of galactomannan and constitutes the endosperm of guar seeds that acts as a reserve polysaccharide for germination. Due to its molecular structure and physical properties, this biopolymer has been considered as one of the most important and widely used gums in industry. However, for many of these applications this (hemi-) cellulosic structure needs to be modified or (partially) depolymerized in order to customize and improve its physicochemical properties. In this study, transcriptome, exoproteome and enzyme activity analyses were employed to decipher the complete enzymatic arsenal for guar gum depolymerization by Aspergillus niger. This multi-omic analysis revealed a set of 46 genes encoding carbohydrate-active enzymes (CAZymes) responding to the presence of guar gum, including CAZymes not only with preferred activity towards galactomannan, but also towards (arabino-) xylan, cellulose, starch and pectin, likely due to trace components in guar gum. This demonstrates that the purity of substrates has a strong effect on the resulting enzyme mixture produced by A. niger and probably by other fungi as well, which has significant implications for the commercial production of fungal enzyme cocktails.
  • Federico, Antonio; Serra, Angela; Ha, My Kieu; Kohonen, Pekka; Choi, Jang-Sik; Liampa, Irene; Nymark, Penny; Sanabria, Natasha; Cattelani, Luca; Fratello, Michele; Kinaret, Pia Anneli Sofia; Jagiello, Karolina; Puzyn, Tomasz; Melagraki, Georgia; Gulumian, Mary; Afantitis, Antreas; Sarimveis, Haralambos; Yoon, Tae-Hyun; Grafström, Roland; Greco, Dario (2020)
    Preprocessing of transcriptomics data plays a pivotal role in the development of toxicogenomics-driven tools for chemical toxicity assessment. The generation and exploitation of large volumes of molecular profiles, following an appropriate experimental design, allows the employment of toxicogenomics (TGx) approaches for a thorough characterisation of the mechanism of action (MOA) of different compounds. To date, a plethora of data preprocessing methodologies have been suggested. However, in most cases, building the optimal analytical workflow is not straightforward. A careful selection of the right tools must be carried out, since it will affect the downstream analyses and modelling approaches. Transcriptomics data preprocessing spans across multiple steps such as quality check, filtering, normalization, batch effect detection and correction. Currently, there is a lack of standard guidelines for data preprocessing in the TGx field. Defining the optimal tools and procedures to be employed in the transcriptomics data preprocessing will lead to the generation of homogeneous and unbiased data, allowing the development of more reliable, robust and accurate predictive models. In this review, we outline methods for the preprocessing of three main transcriptomic technologies including microarray, bulk RNA-Sequencing (RNA-Seq), and single cell RNA-Sequencing (scRNA-Seq). Moreover, we discuss the most common methods for the identification of differentially expressed genes and to perform a functional enrichment analysis. This review is the second part of a three-article series on Transcriptomics in Toxicogenomics.
  • Barreiro, Karina; Dwivedi, Om Prakash; Valkonen, Sami; Groop, Per-Henrik; Tuomi, Tiinamaija; Holthofer, Harry; Rannikko, Antti; Yliperttula, Marjo; Siljander, Pia; Laitinen, Saara; Serkkola, Elina; af Hällström, Taija; Forsblom, Carol; Groop, Leif; Puhka, Maija (2021)
    Urinary extracellular vesicles (uEV) are a topical source of non-invasive biomarkers for health and diseases of the urogenital system. However, several challenges have become evident in the standardization of uEV pipelines from collection of urine to biomarker analysis. Here, we studied the effect of pre-analytical variables and developed means of quality control for uEV isolates to be used in transcriptomic biomarker research. We included urine samples from healthy controls and individuals with type 1 or type 2 diabetes and normo-, micro- or macroalbuminuria and isolated uEV by ultracentrifugation. We studied the effect of storage temperature (-20 degrees C vs. -80 degrees C), time (up to 4 years) and storage format (urine or isolated uEV) on quality of uEV by nanoparticle tracking analysis, electron microscopy, Western blotting and qPCR. Urinary EV RNA was compared in terms of quantity, quality, and by mRNA or miRNA sequencing. To study the stability of miRNA levels in samples isolated by different methods, we created and tested a list of miRNAs commonly enriched in uEV isolates. uEV and their transcriptome were preserved in urine or as isolated uEV even after long-term storage at -80 degrees C. However, storage at -20 degrees C degraded particularly the GC-rich part of the transcriptome and EV protein markers. Transcriptome was preserved in RNA samples extracted with and without DNAse, but read distributions still showed some differences in e.g. intergenic and intronic reads. MiRNAs commonly enriched in uEV isolates were stable and concordant between different EV isolation methods. Analysis of never frozen uEV helped to identify surface characteristics of particles by EM. In addition to uEV, qPCR assays demonstrated that uEV isolates commonly contained polyoma viruses. Based on our results, we present recommendations how to store and handle uEV isolates for transcriptomics studies that may help to expedite standardization of the EV biomarker field.