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  • 1. Acero Sanchez, Josep Ll.
    et al.
    Joda, Hamdi
    Henry, Olivier Y. F.
    Solnestam, Beata W.
    Kvastad, Linda
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sahlén, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Laddach, Nadja
    Ramakrishnan, Dheeraj
    Riley, Ian
    Schwind, Carmen
    Latta, Daniel
    O'Sullivan, Ciara K.
    Electrochemical Genetic Profiling of Single Cancer Cells2017In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 89, no 6, p. 3378-3385Article in journal (Refereed)
    Abstract [en]

    Recent understandings in the development and spread of cancer have led to the realization of novel single cell analysis platforms focused on circulating tumor cells (CTCs). A simple, rapid, and inexpensive analytical platform capable of providing genetic information on these rare cells is highly desirable to support clinicians and researchers alike to either support the selection or adjustment of therapy or provide fundamental insights into cell function and cancer progression mechanisms. We report on the genetic profiling of single cancer cells, exploiting a combination of multiplex ligation-dependent probe amplification (MLPA) and electrochemical detection. Cells were isolated using laser capture and lysed, and the mRNA was extracted and transcribed into DNA. Seven markers were amplified by MLPA, which allows for the simultaneous amplification of multiple targets with a single primer pair, using MLPA probes containing unique barcode sequences. Capture probes complementary to each of these barcode sequences were immobilized on a printed circuit board (PCB) manufactured electrode array and exposed to single-stranded MLPA products and subsequently to a single stranded DNA reporter probe bearing a HRP molecule, followed by substrate addition and fast electrochemical pulse amperometric detection. We present asimple, rapid, flexible, and inexpensive approach for the simultaneous quantification of multiple breast cancer related mRNA markers, with single tumor cell sensitivity.

  • 2.
    Akan, Pelin
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Alexeyenko, Andrey
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Costea, Paul Igor
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hedberg, Lilia
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Werne Solnestam, Beata
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundin, Sverker
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hallman, Jimmie
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Comprehensive analysis of the genome transcriptome and proteome landscapes of three tumor cell lines2012In: Genome Medicine, ISSN 1756-994X, E-ISSN 1756-994X, Vol. 4, p. 86-Article in journal (Refereed)
    Abstract [en]

    We here present a comparative genome, transcriptome and functional network analysis of three human cancer cell lines (A431, U251MG and U2OS), and investigate their relation to protein expression. Gene copy numbers significantly influenced corresponding transcript levels; their effect on protein levels was less pronounced. We focused on genes with altered mRNA and/or protein levels to identify those active in tumor maintenance. We provide comprehensive information for the three genomes and demonstrate the advantage of integrative analysis for identifying tumor-related genes amidst numerous background mutations by relating genomic variation to expression/protein abundance data and use gene networks to reveal implicated pathways.

  • 3.
    Akan, Pelin
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO).
    Stranneheim, Henrik
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO).
    Lexow, Preben
    LingVitae, Oslo.
    Lundeberg, Joakim
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO).
    Design and assessment of binary DNA for nanopore sequencing2010In: Genome biology, ISSN 1474-760X, Vol. 11, p. P4-Article in journal (Other academic)
  • 4.
    Anil, Anandashankar
    et al.
    KTH, School of Biotechnology (BIO). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Spalinskas, Rapolas
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO).
    Åkerborg, Örjan
    KTH, School of Biotechnology (BIO). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sahlén, Pelin
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO).
    HiCapTools: a software suite for probe design and proximity detection for targeted chromosome conformation capture applications2018In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 34, no 4, p. 675-677Article in journal (Refereed)
    Abstract [en]

    Folding of eukaryotic genomes within nuclear space enables physical and functional contacts between regions that are otherwise kilobases away in sequence space. Targeted chromosome conformation capture methods (T2C, chi-C and HiCap) are capable of informing genomic contacts for a subset of regions targeted by probes. We here present HiCapTools, a software package that can design sequence capture probes for targeted chromosome capture applications and analyse sequencing output to detect proximities involving targeted fragments. Two probes are designed for each feature while avoiding repeat elements and non-unique regions. The data analysis suite processes alignment files to report genomic proximities for each feature at restriction fragment level and is isoform-aware for gene features. Statistical significance of contact frequencies is evaluated using an empirically derived background distribution. Targeted chromosome conformation capture applications are invaluable for locating target genes of disease-associated variants found by genome-wide association studies. Hence, we believe our software suite will prove to be useful for a wider user base within clinical and functional applications.

  • 5.
    Costea, Paul Igor
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Akan, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    TagGD: Fast and Accurate Software for DNA Tag Generation and Demultiplexing2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 3, p. e57521-Article in journal (Refereed)
    Abstract [en]

    Multiplexing is of vital importance for utilizing the full potential of next generation sequencing technologies. We here report TagGD (DNA-based Tag Generator and Demultiplexor), a fully-customisable, fast and accurate software package that can generate thousands of barcodes satisfying user-defined constraints and can guarantee full demultiplexing accuracy. The barcodes are designed to minimise their interference with the experiment. Insertion, deletion and substitution events are considered when designing and demultiplexing barcodes. 20,000 barcodes of length 18 were designed in 5 minutes and 2 million barcoded Illumina HiSeq-like reads generated with an error rate of 2% were demultiplexed with full accuracy in 5 minutes. We believe that our software meets a central demand in the current high-throughput biology and can be utilised in any field with ample sample abundance. The software is available on GitHub (https://github.com/pelinakan/UBD.git).

  • 6. Hu, M.
    et al.
    Ayub, Q.
    Guerra-Assunção, J. A.
    Long, Q.
    Ning, Z.
    Huang, N.
    Romero, I. G.
    Mamanova, L.
    Akan, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Liu, X.
    Coffey, A. J.
    Turner, D. J.
    Swerdlow, H.
    Burton, J.
    Quail, M. A.
    Conrad, D. F.
    Enright, A. J.
    Tyler-Smith, C.
    Xue, Y.
    Exploration of signals of positive selection derived from genotype-based human genome scans using re-sequencing data2012In: Human Genetics, ISSN 0340-6717, E-ISSN 1432-1203, Vol. 131, no 5, p. 665-674Article in journal (Refereed)
    Abstract [en]

    We have investigated whether regions of the genome showing signs of positive selection in scans based on haplotype structure also show evidence of positive selection when sequence-based tests are applied, whether the target of selection can be localized more precisely, and whether such extra evidence can lead to increased biological insights. We used two tools: simulations under neutrality or selection, and experimental investigation of two regions identified by the HapMap2 project as putatively selected in human populations. Simulations suggested that neutral and selected regions should be readily distinguished and that it should be possible to localize the selected variant to within 40 kb at least half of the time. Re-sequencing of two ∼300 kb regions (chr4:158Mb and chr10:22Mb) lacking known targets of selection in HapMap CHB individuals provided strong evidence for positive selection within each and suggested the micro-RNA gene hsa-miR-548c as the best candidate target in one region, and changes in regulation of the sperm protein gene SPAG6 in the other.

  • 7.
    Kvastad, Linda
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Werne Solnestam, Beata
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Johansson, Erik
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nygren, A. O.
    Laddach, N.
    Sahlén, Pelin
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vickovic, Sanja
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Bendigtsen, S. C.
    Aaserud, M.
    Floer, L.
    Borgen, E.
    Schwind, C.
    Himmelreich, R.
    Latta, D.
    Lundeberg, Joakim
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Single cell analysis of cancer cells using an improved RT-MLPA method has potential for cancer diagnosis and monitoring2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 16519Article in journal (Refereed)
    Abstract [en]

    Single cell analysis techniques have great potential in the cancer genomics feld. The detection and characterization of circulating tumour cells are important for identifying metastatic disease at an early stage and monitoring it. This protocol is based on transcript profiling using Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification (RT-MLPA), which is a specific method for simultaneous detection of multiple mRNA transcripts. Because of the small amount of (circulating) tumour cells, a pre-amplification reaction is performed after reverse transcription to generate a sufficient number of target molecules for the MLPA reaction. We designed a highly sensitive method for detecting and quantifying a panel of seven genes whose expression patterns are associated with breast cancer, and optimized the method for single cell analysis. For detection we used a fluorescence-dependent semi-quantitative method involving hybridization of unique barcodes to an array. We evaluated the method using three human breast cancer cell lines and identified specific gene expression profiles for each line. Furthermore, we applied the method to single cells and confirmed the heterogeneity of a cell population. Successful gene detection from cancer cells in human blood from metastatic breast cancer patients supports the use of RT-MLPA as a diagnostic tool for cancer genomics.

  • 8.
    Sahlén, Pelin
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Abdullayev, Ilgar
    Ramsköld, Daniel
    Matskova, Liudmila
    Rilakovic, Nemanja
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lötstedt, Britta
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Albert, Thomas J.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sandberg, Rickard
    Genome-wide mapping of promoter-anchored interactions with close to single-enhancer resolution2015In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 16, article id 156Article in journal (Refereed)
    Abstract [en]

    Although the locations of promoters and enhancers have been identified in several cell types, we still have limited information on their connectivity. We developed HiCap, which combines a 4-cutter restriction enzyme Hi-C with sequence capture of promoter regions. Applying the method to mouse embryonic stem cells, we identified promoter-anchored interactions involving 15,905 promoters and 71,984 distal regions. The distal regions were enriched for enhancer marks and transcription, and had a mean fragment size of only 699 bp - close to single-enhancer resolution. High-resolution maps of promoter-anchored interactions with HiCap will be important for detailed characterizations of chromatin interaction landscapes.

  • 9. Shirley, B. C.
    et al.
    Mucaki, E. J.
    Whitehead, T.
    Costea, Paul Igor
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Akan, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rogan, P. K.
    Interpretation, Stratification and Evidence for Sequence Variants Affecting mRNA Splicing in Complete Human Genome Sequences2013In: Genomics, Proteomics and Bioinformatics, ISSN 1672-0229, Vol. 11, no 2, p. 77-85Article in journal (Refereed)
    Abstract [en]

    Information theory-based methods have been shown to be sensitive and specific for predicting and quantifying the effects of non-coding mutations in Mendelian diseases. We present the Shannon pipeline software for genome-scale mutation analysis and provide evidence that the software predicts variants affecting mRNA splicing. Individual information contents (in bits) of reference and variant splice sites are compared and significant differences are annotated and prioritized. The software has been implemented for CLC-Bio Genomics platform. Annotation indicates the context of novel mutations as well as common and rare SNPs with splicing effects. Potential natural and cryptic mRNA splicing variants are identified, and null mutations are distinguished from leaky mutations. Mutations and rare SNPs were predicted in genomes of three cancer cell lines (U2OS, U251 and A431), which were supported by expression analyses. After filtering, tractable numbers of potentially deleterious variants are predicted by the software, suitable for further laboratory investigation. In these cell lines, novel functional variants comprised 6-17 inactivating mutations, 1-5 leaky mutations and 6-13 cryptic splicing mutations. Predicted effects were validated by RNA-seq analysis of the three aforementioned cancer cell lines, and expression microarray analysis of SNPs in HapMap cell lines.

  • 10.
    Solnestam, Beata W.
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kvastad, Linda
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Johansson, Elin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nygren, A. O.
    Laddach, N.
    Sahlén, Pellin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Single cell analysis of cancer cells using an improved RT-MLPA method has potential for cancer diagnosis and monitoringManuscript (preprint) (Other academic)
  • 11.
    Stahl, Patrik L.
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Salmen, Fredrik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundmark, Anna
    KTH, School of Biotechnology (BIO), Gene Technology.
    Navarro, Jose Fernandez
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Magnusson, Jens
    Giacomello, Stefania
    KTH, School of Biotechnology (BIO), Gene Technology.
    Asp, Michaela
    Westholm, Jakub O.
    Huss, Mikael
    Mollbrink, Annelie
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Linnarsson, Sten
    Codeluppi, Simone
    Borg, Ake
    Ponten, Fredrik
    Costea, Paul Igor
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sahlen, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Mulder, Jan
    Bergmann, Olaf
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Frisen, Jonas
    Visualization and analysis of gene expression in tissue sections by spatial transcriptomics2016In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 353, no 6294, p. 78-82Article in journal (Refereed)
    Abstract [en]

    Analysis of the pattern of proteins or messenger RNAs (mRNAs) in histological tissue sections is a cornerstone in biomedical research and diagnostics. This typically involves the visualization of a few proteins or expressed genes at a time. We have devised a strategy, which we call "spatial transcriptomics," that allows visualization and quantitative analysis of the transcriptome with spatial resolution in individual tissue sections. By positioning histological sections on arrayed reverse transcription primers with unique positional barcodes, we demonstrate high-quality RNA-sequencing data with maintained two-dimensional positional information from the mouse brain and human breast cancer. Spatial transcriptomics provides quantitative gene expression data and visualization of the distribution of mRNAs within tissue sections and enables novel types of bioinformatics analyses, valuable in research and diagnostics.

  • 12.
    Stranneheim, Henrik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Werne Solnestam, Beata
    KTH, School of Biotechnology (BIO), Gene Technology.
    Akan, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Transcript nuclear retention effects quantification of gene expression levelsManuscript (preprint) (Other academic)
    Abstract [en]

    The majority of published differential gene expression studies have used RNA isolated from whole cell extracts (total RNA), overlooking the potential impact of including the nuclear transcriptome in the analyses. It has not been firmly established that the contribution of nuclear RNA is negligible or how the inclusion of it affects quantification of gene expression. Previous studies have estimated that the nuclear transcriptome is five to ten times more complex than the cytoplasmic [1]. Hence, RNA purified solely from the cytoplasm should have fewer unique transcripts, resulting in more sequence counts per transcript and resulting in increased power to detect remaining transcripts. In this study, cytoplasmic and total mRNA have been prepared from three human cell‐lines and sequenced using massive parallel sequencing. The resulting sequence data was analyzed regarding the effect of number of biological replicates, read length and transcripts fractionation on calling differentially detected genes. In addition, the impact of length and secondary structure of mRNAs un‐translated regions (UTRs), and coding sequence length on nucleus to cytoplasm transportation rates of mRNAs was studied. We observe that the number of differentially detected genes was not significantly increased by adding more than three biological replicates or by increasing the sequence read length > 35bp. More differentially detected genes were found in the cytoplasmic RNA compared to the total RNA and a nuclear retention effect was observed for transcripts with long and structured 5’‐ and 3’‐UTR or long protein coding sequences.

  • 13. Sánchez, J. L. A.
    et al.
    Henry, O. Y. F.
    Joda, H.
    Solnestam, Beata Werne
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kvastad, Linda
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Johansson, Erik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Akan, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lladach, N.
    Ramakrishnan, D.
    Riley, I.
    O'Sullivan, C. K.
    Multiplex PCB-based electrochemical detection of cancer biomarkers using MLPA-barcode approach2016In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 82, p. 224-232Article in journal (Refereed)
    Abstract [en]

    Asymmetric multiplex ligation-dependent probe amplification (MLPA) was developed for the amplification of seven breast cancer related mRNA markers and the MLPA products were electrochemically detected via hybridization. Seven breast cancer genetic markers were amplified by means of the MLPA reaction, which allows for multiplex amplification of multiple targets with a single primer pair. Novel synthetic MLPA probes were designed to include a unique barcode sequence in each amplified gene. Capture probes complementary to each of the barcode sequences were immobilized on each electrode of a low-cost electrode microarray manufactured on standard printed circuit board (PCB) substrates. The functionalised electrodes were exposed to the single-stranded MLPA products and following hybridization, a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the amplified strand completed the genocomplex, which was electrochemically detected following substrate addition. The electrode arrays fabricated using PCB technology exhibited an excellent electrochemical performance, equivalent to planar photolithographically-fabricated gold electrodes, but at a vastly reduced cost (>50 times lower per array). The optimised system was demonstrated to be highly specific with negligible cross-reactivity allowing the simultaneous detection of the seven mRNA markers, with limits of detections as low as 25 pM. This approach provides a novel strategy for the genetic profiling of tumour cells via integrated "amplification-to-detection".

  • 14. Tapia-Paez, I.
    et al.
    Asad, S.
    Taylan, F.
    Spalinskas, Rapolas
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Anandashankar, A.
    Nordenskjold, M.
    Wahlgren, C. F.
    Sahlén, Pelin
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Bradley, M.
    Studies of keratinocyte-specific regulatory interactions by three-dimensional mapping with a focus on atopic dermatitis2018In: British Journal of Dermatology, ISSN 0007-0963, E-ISSN 1365-2133, Vol. 179, no 1, p. E33-E33Article in journal (Refereed)
  • 15.
    Werne Solnestam, Beata
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Stranneheim, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hällman, Jimmie
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Käller, Max
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Akan, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Comparison of total and cytoplasmic mRNA reveals global regulation by nuclear retention and miRNAs2012In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 13, no 1, p. 574-Article in journal (Refereed)
    Abstract [en]

    Background: The majority of published gene-expression studies have used RNA isolated from whole cells, overlooking the potential impact of including nuclear transcriptome in the analyses. In this study, mRNA fractions from the cytoplasm and from whole cells (total RNA) were prepared from three human cell lines and sequenced using massive parallel sequencing. Results: For all three cell lines, of about 15000 detected genes approximately 400 to 1400 genes were detected in different amounts in the cytoplasmic and total RNA fractions. Transcripts detected at higher levels in the total RNA fraction had longer coding sequences and higher number of miRNA target sites. Transcripts detected at higher levels in the cytoplasmic fraction were shorter or contained shorter untranslated regions. Nuclear retention of transcripts and mRNA degradation via miRNA pathway might contribute to this differential detection of genes. The consequence of the differential detection was further investigated by comparison to proteomics data. Interestingly, the expression profiles of cytoplasmic and total RNA correlated equally well with protein abundance levels indicating regulation at a higher level. Conclusions: We conclude that expression levels derived from the total RNA fraction be regarded as an appropriate estimate of the amount of mRNAs present in a given cell population, independent of the coding sequence length or UTRs.

  • 16.
    Zhang, Miao
    et al.
    KTH, School of Information and Communication Technology (ICT), Materials- and Nano Physics, Material Physics, MF.
    Schmidt, Torsten
    KTH, School of Information and Communication Technology (ICT), Materials- and Nano Physics, Material Physics, MF.
    Jemt, Anders
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sahlén, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sychugov, Ilya
    KTH, School of Information and Communication Technology (ICT), Materials- and Nano Physics, Material Physics, MF.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Linnros, Jan
    KTH, School of Information and Communication Technology (ICT), Materials- and Nano Physics, Material Physics, MF.
    Nanopore arrays in a silicon membrane for parallel single-molecule detection: DNA translocation2015In: Nanotechnology, ISSN 0957-4484, E-ISSN 1361-6528, Vol. 26, no 31, article id 314002Article in journal (Refereed)
    Abstract [en]

    Optical nanopore sensing offers great potential in single-molecule detection, genotyping, or DNA sequencing for high-throughput applications. However, one of the bottle-necks for fluorophore-based biomolecule sensing is the lack of an optically optimized membrane with a large array of nanopores, which has large pore-to-pore distance, small variation in pore size and low background photoluminescence (PL). Here, we demonstrate parallel detection of single-fluorophore-labeled DNA strands (450 bps) translocating through an array of silicon nanopores that fulfills the above-mentioned requirements for optical sensing. The nanopore array was fabricated using electron beam lithography and anisotropic etching followed by electrochemical etching resulting in pore diameters down to similar to 7 nm. The DNA translocation measurements were performed in a conventional wide-field microscope tailored for effective background PL control. The individual nanopore diameter was found to have a substantial effect on the translocation velocity, where smaller openings slow the translocation enough for the event to be clearly detectable in the fluorescence. Our results demonstrate that a uniform silicon nanopore array combined with wide-field optical detection is a promising alternative with which to realize massively-parallel single-molecule detection.

  • 17.
    Zhang, Wensheng
    et al.
    Soochow Univ, Cam Su Genom Resource Ctr, Suzhou 215123, Peoples R China.;Wellcome Sanger Inst, Hinxton CB10 1SA, England..
    Chronis, Constantinos
    Univ Calif Los Angeles, David Geffen Sch Med, Dept Biol & Chem, Los Angeles, CA 90095 USA.;Univ Calif Los Angeles, Eli & Edythe Broad Ctr Regenerat Med & Stem Cell, Los Angeles, CA USA.;Univ Calif Los Angeles, Jonsson Comprehens Canc Ctr, Bioinformat Program, Los Angeles, CA 90024 USA.;Univ Calif Los Angeles, Mol Biol Inst, Los Angeles, CA 90095 USA..
    Chen, Xi
    Wellcome Sanger Inst, Hinxton CB10 1SA, England..
    Zhang, Heyao
    Soochow Univ, Cam Su Genom Resource Ctr, Suzhou 215123, Peoples R China..
    Spalinskas, Rapolas
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Pardo, Mercedes
    Chester Beatty Labs, Inst Canc Res, London, England..
    Chen, Liangliang
    Soochow Univ, Cam Su Genom Resource Ctr, Suzhou 215123, Peoples R China..
    Wu, Guangming
    Max Planck Inst Mol Biomed, Dept Cell & Dev Biol, Rontgenstr 20, D-48149 Munster, Germany..
    Zhu, Zhexin
    Wellcome Sanger Inst, Hinxton CB10 1SA, England..
    Yu, Yong
    Wellcome Sanger Inst, Hinxton CB10 1SA, England..
    Yu, Lu
    Chester Beatty Labs, Inst Canc Res, London, England..
    Choudhary, Jyoti
    Chester Beatty Labs, Inst Canc Res, London, England..
    Nichols, Jennifer
    Univ Cambridge, Wellcome Trust Med Res Council, Stem Cell Inst, Tennis Court Rd, Cambridge CB2 1QR, England..
    Parast, Mana M.
    Univ Calif San Diego, Dept Pathol, La Jolla, CA 92093 USA.;Univ Calif San Diego, Sanford Consortium Regenerat Med, La Jolla, CA 92093 USA..
    Greber, Boris
    Max Planck Inst Mol Biomed, Dept Cell & Dev Biol, Rontgenstr 20, D-48149 Munster, Germany..
    Sahlén, Pelin
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Plath, Kathrin
    Univ Calif Los Angeles, David Geffen Sch Med, Dept Biol & Chem, Los Angeles, CA 90095 USA.;Univ Calif Los Angeles, Eli & Edythe Broad Ctr Regenerat Med & Stem Cell, Los Angeles, CA USA.;Univ Calif Los Angeles, Jonsson Comprehens Canc Ctr, Bioinformat Program, Los Angeles, CA 90024 USA.;Univ Calif Los Angeles, Mol Biol Inst, Los Angeles, CA 90095 USA..
    The BAF and PRC2 Complex Subunits Dpf2 and Eed Antagonistically Converge on Tbx3 to Control ESC Differentiation2019In: Cell Stem Cell, ISSN 1934-5909, E-ISSN 1875-9777, Vol. 24, no 1, p. 138-+Article in journal (Refereed)
    Abstract [en]

    BAF complexes are composed of different subunits with varying functional and developmental roles, although many subunits have not been examined in depth. Here we show that the Baf45 subunit Dpf2 maintains pluripotency and ESC differentiation potential. Dpf2 co-occupies enhancers with Oct4, Sox2, p300, and the BAF subunit Brg1, and deleting Dpf2 perturbs ESC self-renewal, induces repression of Tbx3, and impairs mesendodermal differentiation without dramatically altering Brg1 localization. Mesendodermal differentiation can be rescued by restoring Tbx3 expression, whose distal enhancer is positively regulated by Dpf2-dependent H3K27ac maintenance and recruitment of pluripotency TFs and Brg1. In contrast, the PRC2 subunit Eed binds an intragenic Tbx3 enhancer to oppose Dpf2-dependent Tbx3 expression and mesendodermal differentiation. The PRC2 subunit Ezh2 likewise opposes Dpf2-dependent differentiation through a distinct mechanism involving Nanog repression. Together, these findings delineate distinct mechanistic roles for specific BAF and PRC2 subunits during ESC differentiation.

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