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Scott W. Altmann - One of the best experts on this subject based on the ideXlab platform.
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mRNA and 18S RNA coapplication reverse transcription for quantitative gene expression analysis
Analytical Biochemistry, 2005Co-Authors: Scott W. AltmannAbstract:Fluorescence-based reverse transcription real-time quantitative polymerase chain reaction (RT–QPCR) is a highly sensitive method for the detection and quantitation of mRNA. To control and correct for sample variability, some common housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), -actin, and ubiquitin are often used as endogenous standards. Other inteRNAl calibrators such as 18S–ribosomal RNA (18S–RNA) have also been used, but further methodological concerns arise given that ribosomal RNA lacks the 3 poly-A tail typically associated with messenger RNA. To take advantage of the constant expression levels of 18S–RNA and the precision of oligo-(dT) primed Wrst-strand synthesis, we have developed a method that combines oligo-(dT) with an 18S–RNA-speciWc primer in the initial reverse transcription (RT) reaction. This strategy, termed coapplication reverse transcription (Co–RT), allows for the analysis of multiple target genes with the advantages of 18S–RNA normalization from a single RT reaction. In this article, we describe Co–RT and present tissue distribution and expression level analysis of several target genes using this method. Co–RT provides increased sensitivity and higher accuracy than do the standard random primed RT methods.
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mRNA and 18S-RNA coapplication-reverse transcription for quantitative gene expression analysis
Analytical Biochemistry, 2005Co-Authors: Li Ji Zhu, Scott W. AltmannAbstract:Fluorescence-based reverse transcription real-time quantitative polymerase chain reaction (RT-QPCR) is a highly sensitive method for the detection and quantitation of mRNA. To control and correct for sample variability, some common housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin, and ubiquitin are often used as endogenous standards. Other inteRNAl calibrators such as 18S-ribosomal RNA (18S-RNA) have also been used, but further methodological concerns arise given that ribosomal RNA lacks the 3′ poly-A tail typically associated with messenger RNA. To take advantage of the constant expression levels of 18S-RNA and the precision of oligo-(dT) primed first-strand synthesis, we have developed a method that combines oligo-(dT) with an 18S-RNA-specific primer in the initial reverse transcription (RT) reaction. This strategy, termed coapplication reverse transcription (Co-RT), allows for the analysis of multiple target genes with the advantages of 18S-RNA normalization from a single RT reaction. In this article, we describe Co-RT and present tissue distribution and expression level analysis of several target genes using this method. Co-RT provides increased sensitivity and higher accuracy than do the standard random primed RT methods. © 2005 Elsevier Inc. All rights reserved.
Karlheinz Schleifer - One of the best experts on this subject based on the ideXlab platform.
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identification of rumen ciliates using small subunit ribosomal RNA 18S rRNA targeted oligonucleotide probes and fluorescence in situ hybridization fish
Journal of Eukaryotic Microbiology, 2005Co-Authors: J Fried, S Weber, Wolfgang Ludwig, Neil R Mcewan, J C Newbold, T Michalowski, Nadine A Thomas, S Kisidayova, Johannes H P Hackstein, Karlheinz SchleiferAbstract:Up to half of the biomass in the rumen can be represented by ciliates, which play an important role in the digestion processes of their hosts. In the literature, very little information can be found on determination of the diversity of complex rumen ciliate communities. The identification of these fast moving protists is mainly based on live observations and comparisons of their highly variable cell morphologies. It makes accurate identification and quantification of rumen ciliates very difficult if not impossible. The development of fluorescence in situ hybridization (FISH), established as a technique to identify prokaryotes and eukaryotes using ribosomal RNA-targeted fluorescently labeled oligonucleotide probes, is a promising approach to identify and investigate rumen ciliates. The present study, part of CIMES (ciliates as monitors for environmental safety), a project sponsored by the European Commission, shows the problems of applying FISH on rumen ciliates and how to solve them. Tests resulted in a new protocol, which recommends para-formaldehyde and formaldehyde at 1–2% final concentration to preserve the ciliates before applying FISH. Furthermore, seven new oligonucleotide probes could be developed and successfully be tested to identify different rumen ciliate taxa of the order Entodiniomorphida (class Litostomatea) by applying FISH. It is also shown, how FISH together with confocal laser scanning microscopy can improve analyses of ciliate cell morphologies. Thanks to Peter Pristas, Peter Javorsky, Ralf Einspanier, Susanne Ulbrich (providing rumen samples), Seung Yeo Moon-van der Staay, Georg van der Staay (providing unpublished 18S-rDNA sequences), the European Commission (financial support, project QLK3-CT-2002-02151).
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Identification of rumen ciliates using small subunit ribosomal RNA (18S‐rRNA)‐targeted oligonucleotide probes and fluorescence in situ hybridization (FISH)
Journal of Eukaryotic Microbiology, 2005Co-Authors: J Fried, S Weber, Wolfgang Ludwig, Neil R Mcewan, J C Newbold, Nadine A Thomas, S Kisidayova, Johannes H P Hackstein, Tadeusz Michałowski, Karlheinz SchleiferAbstract:Up to half of the biomass in the rumen can be represented by ciliates, which play an important role in the digestion processes of their hosts. In the literature, very little information can be found on determination of the diversity of complex rumen ciliate communities. The identification of these fast moving protists is mainly based on live observations and comparisons of their highly variable cell morphologies. It makes accurate identification and quantification of rumen ciliates very difficult if not impossible. The development of fluorescence in situ hybridization (FISH), established as a technique to identify prokaryotes and eukaryotes using ribosomal RNA-targeted fluorescently labeled oligonucleotide probes, is a promising approach to identify and investigate rumen ciliates. The present study, part of CIMES (ciliates as monitors for environmental safety), a project sponsored by the European Commission, shows the problems of applying FISH on rumen ciliates and how to solve them. Tests resulted in a new protocol, which recommends para-formaldehyde and formaldehyde at 1–2% final concentration to preserve the ciliates before applying FISH. Furthermore, seven new oligonucleotide probes could be developed and successfully be tested to identify different rumen ciliate taxa of the order Entodiniomorphida (class Litostomatea) by applying FISH. It is also shown, how FISH together with confocal laser scanning microscopy can improve analyses of ciliate cell morphologies. Thanks to Peter Pristas, Peter Javorsky, Ralf Einspanier, Susanne Ulbrich (providing rumen samples), Seung Yeo Moon-van der Staay, Georg van der Staay (providing unpublished 18S-rDNA sequences), the European Commission (financial support, project QLK3-CT-2002-02151).
Caiqin Sun - One of the best experts on this subject based on the ideXlab platform.
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Molecular phylogenetic studies on an unnamed bovine Babesia sp. based on small subunit ribosomal RNA gene sequences
Veterinary Parasitology, 2005Co-Authors: Jianxun Luo, Dongying Yang, Aihong Liu, Miling Ma, Shengzhi Dang, Hong Yin, Zhijie Liu, Bingyi Lu, Guiquan Guan, Caiqin SunAbstract:The 18S small subunit ribosomal RNA (18S rRNA) gene of an unnamed Babesia species (designated B. U sp.) was sequenced and analyzed in an attempt to distinguish it from other Babesia species in China. The target DNA segment was amplified by polymerase chain reaction (PCR). The PCR product was ligated to the pGEM-T Easy vector for sequencing. It was found that the length of the 18S rRNA gene of all B. U sp. Kashi 1 and B. U sp. Kashi 2 was 1699 bp and 1689 bp. Two phylogenetic trees were, respectively, inferred based on 18S rRNA sequence of the Chinese bovine Babesia isolates and all of Babesia species available in GenBank. The first tree showed that B. U sp. was situated in the branch between B. major Yili and B. bovis Shannxian, and the second tree revealed that B. U sp. was confined to the same group as B. caballi. The percent identity of B. U sp. with other Chinese Babesia species was between 74.2 and 91.8, while the percent identity between two B. U sp. isolates was 99.7. These results demonstrated that this B. U sp. is different from other Babesia species, but that two B. U sp. isolates obtained with nymphal and adultal Hyalomma anatolicum anatolicum tick belong to the same species. © 2005 Elsevier B.V. All rights reserved.
Hua Wen - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus)
Gene, 2013Co-Authors: Chang Geng Yang, Xian-li Wang, Juan Tian, Wei Liu, Fan Wu, Ming Jiang, Hua WenAbstract:Quantitative real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) has been used frequently to study gene expression related to fish immunology. In such studies, a stable reference gene should be selected to correct the expression of the target gene. In this study, seven candidate reference genes (glyceraldehyde-3-phosphate dehydrogenase (GADPH), ubiquitin-conjugating enzyme (UBCE), 18S ribosomal RNA (18S rRNA), beta-2-microglobulin (B2M), elongation factor 1 alpha (EF1A), tubulin alpha chain-like (TUBA) and beta actin (ACTB)), were selected to analyze their stability and normalization in seven tissues (liver, spleen, kidney, brain, heart, muscle and intestine) of Nile tilapia (Oreochromis niloticus) challenged with Streptococcus agalactiae or Streptococcus iniae, respectively. The results showed that all the candidate reference genes exhibited tissue-dependent transcriptional variations. With PBS injection as a control, UBCE was the most stable and suitable single reference gene in the intestine, liver, brain, kidney, and spleen after S. iniae infection, and in the liver, kidney, and spleen after S. agalactiae infection. EF1A was the most suitable in heart and muscle after S. iniae or S. agalactiae infection. GADPH was the most suitable gene in intestine and brain after S. agalactiae infection. In normal conditions, UBCE and 18S rRNA were the most stably expressed genes across the various tissues. These results showed that for RT-qPCR analysis of tilapia, selecting two or more reference genes may be more suitable for cross-tissue analysis of gene expression. © 2013 Elsevier B.V.
Thomas Friedl - One of the best experts on this subject based on the ideXlab platform.
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inferring taxonomic positions and testing genus level assignments in coccoid green lichen algae a phylogenetic analysis of 18S ribosomal RNA sequences from dictyochloropsis reticulata and from members of the genus myrmecia chlorophyta trebouxiophycea
Journal of Phycology, 1995Co-Authors: Thomas FriedlAbstract:Complete nuclear-encoded small-subunit ribosomal RNA (18S rRNA) coding sequences were determined for the coccoid green algae Dictyochloropsis reticulata (Tschermak-Woess) Tschermak-Woess, Myrmecia astigmatica Vinatzer, and M. bisecta Reisigl, to investigate the taxonomic position of Dictyochloropsis Geitler and of the genus Myrmecia Printz. Phylogenies inferred from these data revealed a sister-group relationship between D. reticulata and certain coccoid green algae that lack motile stages (autosporic coccoids) within the order Microthamniales. The monophyletic origin of the Microthamniales, including autosporic coccoids previously classified in the Chlorophyceae, is clearly resolved by the rRNA sequence data. This finding. shows the considerable taxonomic breadth of that order, whose taxonomic position has been unclear so far. A new class, Trebouxiophyceae, is proposed for this group of green algae. Phylogenetic inferences from the rRNA sequences show paraphyly of the genus Myrmecia. The 18S rRNA sequence data suggest that, among taxa that share similar vegetative cell morphologies, the zoospore characters resolve better the actual genus and species boundaries. Within identical zoospore types, the rRNA data allow further resolution of taxonomic relationships. On the basis of the.se findings, I propose that the genus Friedmannia Chantanachat ± Bold be merged into Myrmecia and that only those species be left in the genus Myrmecia that are identical in particular zoospore characters (i.e. those described in detail for M. israeliensis (Chantanachat ± Bold) comb, nov.), namely M. astigmatica, M. biatorellae (Tschermak-Woess ± Ptesst) Petersen, and M. israeliensis. Myrmecia bisecta has to be excluded from Myrmecia; its taxonomic position within the Trebouxiophyceae is unclear.
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assessing the relationships of some coccoid green lichen algae and the microthamniales chlorophyta with 18S ribosomal RNA gene sequence comparisons
Journal of Phycology, 1994Co-Authors: Thomas Friedl, Cornelia ZeltnerAbstract:Eight complete nuclear-encoded small-subunit ribosomal RNA (18S rRNA) gene sequences were determined for four genera of the Microthamniales (Pleurastrophyceae) and for Gloeotilopsis planctonica Iyengar & Philipose (Ulvophyceae, Ulotrichales) to investigate evolutionary relationships within the Microthamniales and the taxonomic position of this order within the green algae. Phylogenies inferred from these data revealed specific relationships at the level of genera and species that disagree with those inferred from vegetative cell morphology but agree with those inferred from motile cell characters. The rRNA phylogenies provide even better resolution than that gained from morphology alone. The coccoid lichen alga Trebouxia spp. is specifically related to other coccoid lichen and soil algae (i.e. Myrmecia biatorellae Boye-Petersen and Friedmannia israelensis Chantanachat & Bold), forming the “Lichen Algae Group,” an evolutionarily distinct lineage within the Microthamniales. Trebouxia is a paraphyletic and Pleurastrum a polyphyletic genus in rRNA phylogenies. In contrast to previous hypotheses based on morphology, Pleurastrum terrestre Fritsch & John is not closely related to Trebouxia but occupies an isolated position within the Microthamniales. The filamentous alga Microthamnion kuetzingianum is not ancestral to coccoid members of the Microthamniales but is closely related with the coccoid Fusochloris perforata (Lee & Bold) Floyd, Watanabe & Deason. The Microthamniales are inferred to be an array of independent lineages that radiate nearly simultaneously and may also include some autosporic coccoid taxa previously classified in the Chlorophyceae. Monophyly of the Microthamniales could not be demonstrated unequivocally. In contrast to a hypothesis based on ultrastructure, the Microthamniales are evolutionarily distinct from the Ulotrichales. The latter are ancestral to the radiation of the Microthamniales and the Chlorophyceae in the rRNA phylogenies.
