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Northern blot

Todd Blevins
Cells have evolved intricate RNA-directed mechanisms that destroy viruses, silence transposons, and regulate gene expression. These nucleic acid surveillance and gene silencing mechanisms rely upon the selective base-pairing of ~19-25 nt small RNAs to complementary RNA targets. This chapter describes northern blot hybridization techniques for the detection of such small RNAs. Blots spiked with synthetic standards are used to illustrate the detection specificity and sensitivity of DNA oligonucleotide probes...
2017: Methods in Molecular Biology
Subir K Nagdas, Virginia P Winfrey, Gary E Olson
Nevertheless, a nonviable sperm population is present in the cauda epididymidis of many species. Degenerating spermatozoa release enzymes that could have detrimental effects on the viability of neighboring cells, and they are source of autoantigens that induce an autoimmune response if they escape the blood-epididymis barrier. Does the epididymis have specialized protective mechanism(s) to segregate the viable sperm population from defective spermatozoa? Previously, we identified a fibrinogen-like protein-2 (fgl2) that specifically binds to and polymerizes into a cocoon-like complex coating defective spermatozoa and sperm fragments...
October 9, 2016: International Journal of Biochemistry & Cell Biology
Ying Xiong, Te Liu, Suwei Wang, Huiying Chi, Chuan Chen, Jin Zheng
The dysfunction of ovarian granulosa cells (OGCs) directly affects the premature ovarian failure (POF). In vivo experiments showed that cyclophosphamide significantly induced mouse ovarian atrophy and proliferation inhibition of OGCs. The expressions of p53, p66Shc and p16 were significantly higher in OGCs of the cyclophosphamide treatment group. MTT assay showed that cyclophosphamide effectively inhibited the proliferation of OGCs in vitro. SA-β-Gal staining showed that the OGCs in the cyclophosphamide treatment group had many senescent cells...
October 8, 2016: Gene
Minoru Kato, Yi-Ying Huang, Mina Matsuo, Yoko Takashina, Kazuyo Sasaki, Yasushi Horai, Aya Juni, Shin-Ichi Kamijo, Kaoru Saigo, Kumiko Ui-Tei, Hajime Tei
RNA interference (RNAi) is a powerful tool for the study of gene function in mammalian systems, including transgenic mice. Here, we report a gene knockdown system based on the human mir-187 precursor. We introduced small interfering RNA (siRNA) sequences against the mouse melanocortin-4 receptor (mMc4r) to alter the targeting of miR-187. The siRNA-expressing cassette was placed under the control of the cytomegalovirus (CMV) early enhancer/chicken β-actin promoter. In vitro, the construct efficiently knocked down the gene expression of a co-transfected mMc4r-expression vector in cultured mammalian cells...
September 30, 2016: Experimental Animals
Chih-Hao Chang, Fu-Chen Hsu, Shu-Chuan Lee, Yih-Shan Lo, Jiun-Da Wang, Jane Shaw, Michael Taliansky, Ban-Yang Chang, Yau-Heiu Hsu, Na-Sheng Lin
RNA trafficking plays pivotal roles in regulating plant development, gene silencing, and adaptation to environmental stress. Satellite RNAs (satRNAs), parasites of viruses, depend on their helper viruses (HVs) for replication, encapsidation, and efficient spread. However, it remains largely unknown how satRNAs interact with viruses and the cellular machinery to undergo trafficking. Here, we show that the P20 protein of Bamboo mosaic potexvirus satRNA (satBaMV) can functionally complement in trans the systemic trafficking of P20-defective satBaMV in infected Nicotiana benthamiana...
October 4, 2016: Plant Cell
Ke Zhang
An important aspect of RNAi-mediated heterochromatin assembly is the production of short interfering RNAs (siRNAs) in cis at the heterochromatic target. These populations of small RNAs (20-30 nt) can be detected via northern blot analysis with radiolabeled RNA probes corresponding to the siRNAs. Most heterochromatin-associated siRNAs originate from both strands of the pericentromeric dg and dh repeats. Thus, production of RNA probes by in vitro transcription utilizes a DNA template with sequences corresponding to either dg or dh repeats...
October 3, 2016: Cold Spring Harbor Protocols
K B Rebijith, R Asokan, H Ranjitha Hande, N K Krishna Kumar
Thrips palmi Karny (Thysanoptera: Thripidae) is the sole vector of Watermelon bud necrosis tospovirus, where the crop loss has been estimated to be around USD 50 million annually. Chemical insecticides are of limited use in the management of T. palmi due to the thigmokinetic behaviour and development of high levels of resistance to insecticides. There is an urgent need to find out an effective futuristic management strategy, where the small RNAs especially microRNAs hold great promise as a key player in the growth and development...
2016: PloS One
Annekathrin Rumlow, Els Keunen, Jan Klein, Philip Pallmann, Anja Riemenschneider, Ann Cuypers, Jutta Papenbrock
Analysis of gene expression is one of the major ways to better understand plant reactions to changes in environmental conditions. The comparison of many different factors influencing plant growth challenges the gene expression analysis for specific gene-targeted experiments, especially with regard to the choice of suitable reference genes. The aim of this study is to compare expression results obtained by Northern blot, semi-quantitative PCR and RT-qPCR, and to identify a reliable set of reference genes for oilseed rape (Brassica napus L...
2016: PloS One
Kimberly R McDermid, Andrei Snyman, Frederick J Verreynne, John P Carroll, Banie L Penzhorn, Michael J Yabsley
African lion ( Panthera leo ) numbers are decreasing rapidly and populations are becoming smaller and more fragmented. Infectious diseases are one of numerous issues threatening free-ranging lion populations, and low-density populations are particularly at risk. We collected data on the prevalence and diversity of viral and parasitic pathogens in a small lion population in eastern Botswana. During 2012 and 2014, blood samples were collected from 59% (n=13) of the adult-subadult lions in the Northern Tuli Game Reserve in eastern Botswana...
September 26, 2016: Journal of Wildlife Diseases
Suk-Hyun Yun, Song Hee Lee, Kum-Kang So, Jung-Mi Kim, Dae-Hyuk Kim
A total of 315 fungal isolates causing green mold disease were collected from contaminated artificial logs and sawdust bags used for cultivating shiitake Lentinula edodes in Korea and were analyzed for the presence of double-stranded RNA (dsRNA). dsRNA, which was purified using dsRNA-specific chromatography and verified by dsRNA-specific RNaseIII digestion, was detected in 32 isolates. The molecular taxonomy of dsRNA-infected isolates indicated that all isolates belonged to the Trichoderma spp.. The number and size of dsRNAs varied among isolates and the band patterns could be categorized into 15 groups...
September 22, 2016: FEMS Microbiology Letters
Arielle Klepper, Francis J Eng, Erin H Doyle, Ahmed El-Shamy, Adeeb H Rahman, M Isabel Fiel, Gonzalo Carrasco Avino, Moonju Lee, Fei Ye, Sasan Roayaie, Meena B Bansal, Margaret R MacDonald, Thomas D Schiano, Andrea D Branch
: Hepatitis C virus (HCV) is unique among RNA viruses in its ability to establish chronic infection in the majority of exposed adults. HCV persists in the liver despite interferon-stimulated gene (ISG) induction; robust induction actually predicts treatment failure and viral persistence. It is unclear which forms of HCV RNA are associated with ISG induction and interferon resistance during natural infections. To thoroughly delineate HCV RNA populations, we developed conditions that fully separate the strands of long double-stranded (ds)RNA and allow the released RNAs to be quantified in reverse transcription/polymerase chain reaction (qRT/PCR) assays...
September 19, 2016: Hepatology: Official Journal of the American Association for the Study of Liver Diseases
Cláudia Gomes, Noemí Palma, Maria J Pons, Ariel Magallón-Tejada, Isabel Sandoval, Carmen Tinco-Valdez, Carlos Gutarra, Juana Del Valle-Mendoza, Joaquim Ruiz, Mayumi Matsuoka
BACKGROUND: Bartonella bacilliformis is the causative agent of Carrion's disease, a neglected illness with mortality rates of 40-85% in the absence of treatment. The lack of a diagnostic technique to overcome misdiagnosis and treat asymptomatic carriers is of note. This study aimed to identify new B. bacilliformis antigenic candidates that could lead to a new diagnostic tool able to be implemented in endemic rural areas. METHODOLOGY/PRINCIPAL FINDINGS: Blood (n = 198) and serum (n = 177) samples were collected in northern Peru...
September 2016: PLoS Neglected Tropical Diseases
Elena Senís, Stefan Mockenhaupt, Daniel Rupp, Tobias Bauer, Nagarajan Paramasivam, Bettina Knapp, Jan Gronych, Stefanie Grosse, Marc P Windisch, Florian Schmidt, Fabian J Theis, Roland Eils, Peter Lichter, Matthias Schlesner, Ralf Bartenschlager, Dirk Grimm
Successful RNAi applications depend on strategies allowing robust and persistent expression of minimal gene silencing triggers without perturbing endogenous gene expression. Here, we propose a novel avenue which is integration of a promoterless shmiRNA, i.e. a shRNA embedded in a micro-RNA (miRNA) scaffold, into an engineered genomic miRNA locus. For proof-of-concept, we used TALE or CRISPR/Cas9 nucleases to site-specifically integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr locus in hepatoma cells, with the aim to obtain cellular clones that are genetically protected against HCV infection...
September 9, 2016: Nucleic Acids Research
Catrin Stutika, Mario Mietzsch, Andreas Gogol-Döring, Stefan Weger, Madlen Sohn, Wei Chen, Regine Heilbronn
Most DNA viruses express small regulatory RNAs, which interfere with viral or cellular gene expression. For adeno-associated virus (AAV), a small ssDNA virus with a complex biphasic life cycle miRNAs or other small regulatory RNAs have not yet been described. This is the first comprehensive Illumina-based RNA-Seq analysis of small RNAs expressed by AAV alone or upon co-infection with helper adenovirus or HSV. Several hotspots of AAV-specific small RNAs were detected mostly close to or within the AAV-ITR and apparently transcribed from the newly identified anti-p5 promoter...
2016: PloS One
Andre Bertran, Marina Ciuffo, Paolo Margaria, Cristina Rosa, Renato Oliveira Resende, Massimo Turina
Polygonum ringspot virus (PolRSV) is a recently characterized tospovirus reported in Italy. Northern blot analyses of PolRSV infections in Nicotiana benthamiana (NB) and tomato plants showed that a viral RNA species with nearly twice the length of the genomic S-RNA accumulated abundantly in the former host, but was not detected in the latter. Additional assays confirmed that biogenesis of this novel RNA species was common to all PolRSV isolates tested and also to an isolate of Tomato spotted wilt virus (TSWV)...
September 6, 2016: Journal of General Virology
Susana Manzano, Zoraida Megías, Cecilia Martínez, Alicia García, Encarnación Aguado, Tarik Chileh, Diego López-Alonso, Federico García-Maroto, Eduard Kejnovský, Jiří Široký, Zdeněk Kubát, Tereza Králová, Boris Vyskot, Manuel Jamilena
Sex determination in Rumex acetosa, a dioecious plant with a complex XY1 Y2 sex chromosome system (females are XX and males are XY1 Y2 ), is not controlled by an active Y chromosome but depends on the ratio between the number of X chromosomes and autosomes. To gain insight into the molecular mechanisms of sex determination, we generated a subtracted cDNA library enriched in genes specifically or predominantly expressed in female floral buds in early stages of development, when sex determination mechanisms come into play...
September 6, 2016: Plant Journal: for Cell and Molecular Biology
Fosheng Li, Lanju Mei, Cheng Zhan, Qiang Mao, Min Yao, Shenghua Wang, Lin Tang, Fang Chen
MicroRNAs (miRNAs) play important roles in nearly every aspect of biology, including physiological, biochemical, developmental and pathological processes. Therefore, a highly sensitive and accurate method of detection of miRNAs has great potential in research on theory and application, such as the clinical approach to medicine, animal and plant production, as well as stress response. Here, we report a strategic method to detect miRNAs from multicellular organisms, which mainly includes liquid hybridization and solid phase detection (LHSPD); it has been verified in various species and is much more sensitive than traditional biotin-labeled Northern blots...
2016: International Journal of Molecular Sciences
Aya Zamoto-Niikura, Shigeru Morikawa, Ken-Ichi Hanaki, Patricia J Holman, Chiaki Ishihara
: U.S. lineage, one of the major clades in the Babesia microti group, is known as a causal agent of human babesiosis mostly in the Northeastern and upper Midwestern United States. This lineage, however, distributes as well throughout the temperate zone of Eurasia with several reported human cases, although convincing evidence of the identity of the specific vector(s) in this area is lacking. Here, the goal was to demonstrate the presence of infectious parasites directly in salivary glands of Ixodes persulcatus, from which U...
September 2, 2016: Applied and Environmental Microbiology
Suneha Goswami, Ranjeet R Kumar, Kavita Dubey, Jyoti P Singh, Sachidanand Tiwari, Ashok Kumar, Shuchi Smita, Dwijesh C Mishra, Sanjeev Kumar, Monendra Grover, Jasdeep C Padaria, Yugal K Kala, Gyanendra P Singh, Himanshu Pathak, Viswanathan Chinnusamy, Anil Rai, Shelly Praveen, Raj D Rai
Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h) wheat cv. HD2985 by suppression subtractive hybridization (SSH). We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sanger's sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs). Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways...
2016: Frontiers in Plant Science
Minshi Wang, Dimitri G Pestov
Assembly of eukaryotic ribosomes is an elaborate biosynthetic process that begins in the nucleolus and requires hundreds of cellular factors. Analysis of rRNA processing has been instrumental for studying the mechanisms of ribosome biogenesis and effects of stress conditions on the molecular milieu of the nucleolus. Here, we describe the quantitative analysis of the steady-state levels of rRNA precursors, applicable to studies in mammalian cells and other organisms. We include protocols for gel electrophoresis and northern blotting of rRNA precursors using procedures optimized for the large size of these RNAs...
2016: Methods in Molecular Biology
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