scholarly journals Fluorophores from fungi

2003 ◽  
Vol 24 (3) ◽  
pp. 12 ◽  
Author(s):  
Duncan Veal ◽  
Philip Bell ◽  
Hayley Brown ◽  
Hung-Yoon Choi ◽  
Peter Karuso
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Dna Microarray ◽  
Differential Display ◽  
Real Time Pcr ◽  
In Situ Hybridisation ◽  
Analytical Techniques ◽  
Fluorescence In Situ Hybridisation

Fluorescence has many advantages over traditional colour and radioactive labels, and is playing an increasingly important role in the most powerful analytical techniques. For example, fluorescence is at the heart of many nucleic acid based diagnostics (e.g. DNA microarray, real time-PCR, fluorescence in situ hybridisation, etc), immunofluorescence assays, defined substrate technologies and differential display proteomics and is gradually replacing or complementing other techniques based on colour or radiolabels.

Assessing the abundance and activity of denitrifying polyphosphate accumulating organisms through molecular and chemical techniques

2010 ◽  
Vol 61 (8) ◽  
pp. 2061-2068 ◽  
Author(s):  
Adrian Oehmen ◽  
Gilda Carvalho ◽  
Filomena Freitas ◽  
Maria AM Reis
Keyword(s):  
In Situ Hybridisation ◽  
Analytical Techniques ◽  
Biological Nutrient Removal ◽  
Future Research ◽  
Fluorescence In Situ Hybridisation ◽  
Batch Tests ◽  
Oxygen Requirements ◽  
Polyphosphate Accumulating Organisms ◽  
Quantitative Fluorescence

Biological nutrient removal (BNR) plants can reduce both carbon and oxygen requirements by increasing the fraction of phosphorus (P) removed by denitrifying polyphosphate accumulating organisms (DPAOs). Contrasting findings have been reported in literature concerning whether or not PAOs and DPAOs are different microorganisms. In this study, quantitative fluorescence in situ hybridisation (FISH) measurements from different EBPR sludges support the hypothesis that PAOs and DPAOs are phyogenetically different. This experimental evidence is discussed within the context of literature findings and suggestions for future research concerning the identity of PAOs and DPAOs are proposed. Further, this paper discusses the different methodologies available for assessing the DPAO fraction through chemical analytical techniques, where the relative fraction estimated is highly dependent on the methodology employed. Thus, we recommend an alteration to previously proposed methods in order to calculate the DPAO fraction through anaerobic-anoxic and anaerobic-aerobic batch tests. This information is expected to be valuable in studies focussed on optimising the amount of phosphorus removal achieved with simultaneous denitrification.

Association of MicroRNA Expression Profiles with Karyotype in Acute Myeloid Leukaemias Revealed by Real-Time PCR and In Situ Hybridisation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 105-105
Author(s):  
Amanda Dixon-McIver ◽  
Phil East ◽  
Charles A. Mein ◽  
Jean-Baptiste Cazier ◽  
Gael Molloy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Real Time ◽  
Mirna Expression ◽  
Real Time Pcr ◽  
Expression Profiles ◽  
In Situ Hybridisation ◽  
Locked Nucleic Acid ◽  
Healthy Donors ◽  
Acute Myeloid

Abstract MicroRNAs (miRNAs) are single stranded non-coding RNAs of ∼ 22 nucleotides involved in gene regulation. Several examples of an association between disrupted expression of miRNAs and cancer have been shown. Using a real-time quantitative PCR, designed to amplify only from the mature miRNA (TaqMan® -MicroRNA Looped-PCR assay, Applied Biosystems), we measured the expression levels of 157 miRNAs in 100 acute myeloid leukaemia (AML) patients representing the spectrum of known karyotypes common in AML, 2 leukaemic cell lines (KG1 and NB4), and the bone marrow from 2 healthy donors. ANOVA analysis using a 5% false discovery rate threshold was performed to identify differentially expressed miRNAs between leukaemia samples and normal bone marrow. MiRNAs specific to karyotype groupings were also identified in the same way. A method was developed to demonstrate the spatial localisation, in situ, of specific miRNA identified in the quantification and confirm the expression of miRNA with relation to karyotype. Commercial locked nucleic acid (LNA) oligonucleotides (Exiqon) were obtained for two miRNAs (miRNA-127 and miRNA-154), as well as for positive and negative control probes. LNAs were labelled with digoxigenin and probes applied to both cytospins and/or trephines of a sample set representative of the different AML subtypes. Detection of hybridisation signals was either by colorimetric or fluorescent reaction and visualised by confocal microscopy. Unsupervised cluster analysis revealed an association of miRNA expression with the karyotype of the samples. Promyelocytic leukaemias (APML) bearing the t(15;17) translocation, show a distinct pattern characterised by the high expression of a subset of 10 miRNAs located in the human 14q32 imprinted domain, including miR-127 and miR-154. ANOVA data analysis revealed the de-regulation of 33 miRNAs across the leukaemic set in respect to bone marrow from healthy donors. Seventeen miRNAs were up-regulated and 16 down-regulated. MiRNAs miR-155, miR-181a, miR-181b, miR-181c, miR-142-5p, miR-221, and miR-222, were among those commonly highly expressed. Down-regulated were miR-26a, miR-34c, and miR-199a. MiRNAs miR-10a and miR-125b showed the highest variability throughout the samples, being associated with specific subgroups. In situ hybridisation analysis of miR-127 and miR-154 confirmed the results obtained by real-time PCR of their expression associated with APML. This study, conducted on about a third of the miRNAs reported in the Sanger database (microrna.sanger.ac.uk), demonstrates the potential for using miRNA expression to sub-classify cancer. The expression analysis of larger number of miRNAs coupled with the in situ hybridisation of leukaemic cells will allow the investigation of miRNA expression on stored samples during the disease course and provide valuable insights into the leukaemogenic process.

227. FSH receptor expression in small human ovarian follicles

2005 ◽  
Vol 17 (9) ◽  
pp. 88
Author(s):  
J. H. Quennell ◽  
J. L. Stanton ◽  
P. R. Hurst
Keyword(s):  
Real Time ◽  
Granulosa Cells ◽  
Real Time Pcr ◽  
In Situ Hybridisation ◽  
Receptor Expression ◽  
Follicle Development ◽  
Fsh Receptor ◽  
Relative Expression ◽  
Receptor Mrna

Follicle-stimulating hormone (FSH) is pivotal in ovarian follicle development; the granulosa cells are the targets of FSH action in the ovary via FSH receptors. Granulosa cell growth and division mark initial follicle recruitment. The acquisition of FSH receptors on granulosa cells is regarded as a key event in hormone responsiveness and consequently follicle development. Due to the low abundance of FSH receptors and low expression of its mRNA it has been difficult to definitively characterise FSH receptor expression patterns. Here, localisation of FSH receptor in different follicle populations has been assessed with in situ hybridisation and real-time PCR of laser microdissected samples. We have used non-radioactive in situ hybridisation to investigate FSH receptor mRNA on a wide range of follicle stages. Biopsies from healthy fertile women (28–33 years) were frozen, embedded and cryosectioned at 10 µm. DIG-labelled RNA probes were designed to detect all splice variants. Hybridised probes were detected with NBT/BCIP in a colorimetric reaction. Secondly, follicles of different morphometric stages were isolated with a laser microscope. RNA extraction, reverse transcription and real-time PCR were used to confirm RNA presence and quantify relative expression. All follicle stages (from primordial to large antral) showed the presence of FSH receptor mRNA in their granulosa cells; sense controls were negative. Observations from real-time PCR indicate FSH receptor mRNA is present in all follicle stages observed and relative expression levels increase over early follicle development. These results challenge the existing doctrine that FSH receptor is absent in the smallest follicles. This suggests initial follicle recruitment may involve gonadotrophins. The use of sensitive molecular techniques will be crucial in elucidating this further.

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

scholarly journals Rapid Multi-Hybridisation FISH Screening for Balanced Porcine Reciprocal Translocations Suggests a Much Higher Abnormality Rate Than Previously Appreciated

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 250
Author(s):  
Rebecca E O’Connor ◽  
Lucas G Kiazim ◽  
Claudia C Rathje ◽  
Rebecca L Jennings ◽  
Darren K Griffin
Keyword(s):  
Semen Analysis ◽  
In Situ Hybridisation ◽  
Economic Loss ◽  
Environmental Damage ◽  
Fluorescence In Situ Hybridisation ◽  
Reciprocal Translocations ◽  
Chromosome Translocations ◽  
Farrowing Rate ◽  
Significant Economic Loss

With demand rising, pigs are the world’s leading source of meat protein; however significant economic loss and environmental damage can be incurred if boars used for artificial insemination (AI) are hypoprolific (sub-fertile). Growing evidence suggests that semen analysis is an unreliable tool for diagnosing hypoprolificacy, with litter size and farrowing rate being more applicable. Once such data are available, however, any affected boar will have been in service for some time, with significant financial and environmental losses incurred. Reciprocal translocations (RTs) are the leading cause of porcine hypoprolificacy, reportedly present in 0.47% of AI boars. Traditional standard karyotyping, however, relies on animal specific expertise and does not detect more subtle (cryptic) translocations. Previously, we reported development of a multiple hybridisation fluorescence in situ hybridisation (FISH) strategy; here, we report on its use in 1641 AI boars. A total of 15 different RTs were identified in 69 boars, with four further animals XX/XY chimeric. Therefore, 4.5% had a chromosome abnormality (4.2% with an RT), a 0.88% incidence. Revisiting cases with both karyotype and FISH information, we reanalysed captured images, asking whether the translocation was detectable by karyotyping alone. The results suggest that chromosome translocations in boars may be significantly under-reported, thereby highlighting the need for pre-emptive screening by this method before a boar enters a breeding programme.

Identifying the etiologic role of Parvovirus B19 in non-immune hydrops fetalis by histopathology, immunohistochemistry and nucleic acid testing: a retrospective study

Open Medicine ◽  
2007 ◽  
Vol 2 (3) ◽  
pp. 271-279 ◽  
Author(s):  
Koray Ergunay ◽  
Gulcin Altinok ◽  
Bora Gurel ◽  
Ahmet Pinar ◽  
Arzu Sungur ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
San Francisco ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Parvovirus B19 ◽  
Etiologic Agent ◽  
Hydrops Fetalis ◽  
Nucleic Acid Detection

AbstractIntrauterine Parvovirus B19 infections may cause fetal anemia, non-immune hydrops fetalis or abortion. This study focuses on the pathogenic role of Parvovirus B19 in non-immune hydrops fetalis at Hacettepe University, a major reference hospital in Turkey. Twenty-two cases of non-immune hydrops fetalis were retrospectively selected out of a total of 431 hydrops fetalis specimens from the Department of Pathology archieves. Paraffine embedded tissue sections from placental and liver tissues from each case were evaluated by histopathology, immunohistochemistry, nested PCR and commercial quantitative Real-time PCR. Viral DNA was detected in placental tissues by Real-time PCR in 2 cases (2/22, 9.1%) where histopathology also revealed changes suggestive of Parvovirus B19 infection. No significant histopathologic changes were observed for the remaining sections. Nested PCR that targets the VP1 region of the viral genome and immunohistochemistry for viral capsid antigens were negative for all cases. As a result, Parvovirus B19 is identified as the etiologic agent for the development of non-immune hydrops fetalis for 9.1% of the cases in Hacettepe University, Turkey. Real-time PCR is observed to be an effective diagnostic tool for nucleic acid detection from paraffine embedded tissues. Part of this study was presented as a poster at XIIIth International Congress of Virology, San Francisco, USA (Abstract V-572).

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