scholarly journals A head-to-head comparison of ribodepletion and polyA selection approaches for C. elegans low input RNA-sequencing libraries

2021 ◽  
Author(s):  
Alec Barrett ◽  
Rebecca McWhirter ◽  
Seth R Taylor ◽  
Alexis Weinreb ◽  
David M Miller ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Noncoding Rnas ◽  
Rrna Gene ◽  
Library Preparation ◽  
C Elegans ◽  
Pcr Duplicates ◽  
Rrna Depletion ◽  
Dissociated Cells ◽  
Probe Set ◽  
Long Genes

Abstract A recent and powerful technique is to obtain transcriptomes from rare cell populations, such as single neurons in C. elegans, by enriching dissociated cells using fluorescent sorting. However, these cell samples often have low yields of RNA that present challenges in library preparation. This can lead to PCR duplicates, noisy gene expression for lowly expressed genes, and other issues that limit endpoint analysis. Further, some common resources, such as sequence specific kits for removing ribosomal RNA, are not optimized for non-mammalian samples. To advance library construction for such challenging samples, we compared two approaches for building RNAseq libraries from less than 10 nanograms of C. elegans RNA: SMARTSeq V4 (Takara), a widely used kit for selecting poly-adenylated transcripts; and SoLo Ovation (Tecan Genomics), a newly developed ribodepletion-based approach. For ribodepletion, we used a custom kit of 200 probes designed to match C. elegans rRNA gene sequences. We found that SoLo Ovation, in combination with our custom C. elegans probe set for rRNA depletion, detects an expanded set of noncoding RNAs, shows reduced noise in lowly expressed genes, and more accurately counts expression of long genes. The approach described here should be broadly useful for similar efforts to analyze transcriptomics when RNA is limiting.

scholarly journals An optimized ribodepletion approach for C. elegans RNA-sequencing libraries

2021 ◽  
Author(s):  
Alec Barrett ◽  
Rebecca McWhirter ◽  
Seth R Taylor ◽  
Alexis Weinreb ◽  
David M Miller ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Noncoding Rnas ◽  
Rrna Gene ◽  
Library Preparation ◽  
C Elegans ◽  
Pcr Duplicates ◽  
Rrna Depletion ◽  
Dissociated Cells ◽  
Probe Set ◽  
Long Genes

ABSTRACTA recent and powerful technique is to obtain transcriptomes from rare cell populations, such as single neurons in C. elegans, by enriching dissociated cells using fluorescent sorting. However, these cell samples often have low yields of RNA that present challenges in library preparation. This can lead to PCR duplicates, noisy gene expression for lowly expressed genes, and other issues that limit endpoint analysis. Further, some common resources, such as sequence specific kits for removing ribosomal RNA, are not optimized for non-mammalian samples. To optimize library construction for such challenging samples, we compared two approaches for building RNAseq libraries from less than 10 nanograms of C. elegans RNA: SMARTSeq V4 (Takara), a widely used kit for selecting poly-adenylated transcripts; and SoLo Ovation (Tecan Genomics), a newly developed ribodepletion-based approach. For ribodepletion, we used a custom kit of 200 probes designed to match C. elegans rRNA gene sequences. We found that SoLo Ovation, in combination with our custom C. elegans probe set for rRNA depletion, detects an expanded set of noncoding RNAs, shows reduced noise in lowly expressed genes, and more accurately counts expression of long genes. The approach described here should be broadly useful for similar efforts to analyze transcriptomics when RNA is limiting.

scholarly journals Comparison of Poly-A+ Selection and rRNA Depletion in Detection of lncRNA in Two Equine Tissues Using RNA-seq

2020 ◽  
Vol 6 (3) ◽  
pp. 32 ◽  
Author(s):  
Anna R. Dahlgren ◽  
Erica Y. Scott ◽  
Tamer Mansour ◽  
Erin N. Hales ◽  
Pablo J. Ross ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Preparation Method ◽  
Parietal Lobe ◽  
Library Preparation ◽  
Rna Seq ◽  
The Past ◽  
Rrna Depletion ◽  
Non Coding Rnas ◽  
Nucleolar Rna ◽  
Library Preparation Method

Long non-coding RNAs (lncRNAs) are untranslated regulatory transcripts longer than 200 nucleotides that can play a role in transcriptional, post-translational, and epigenetic regulation. Traditionally, RNA-sequencing (RNA-seq) libraries have been created by isolating transcriptomic RNA via poly-A+ selection. In the past 10 years, methods to perform ribosomal RNA (rRNA) depletion of total RNA have been developed as an alternative, aiming for better coverage of whole transcriptomic RNA, both polyadenylated and non-polyadenylated transcripts. The purpose of this study was to determine which library preparation method is optimal for lncRNA investigations in the horse. Using liver and cerebral parietal lobe tissues from two healthy Thoroughbred mares, RNA-seq libraries were prepared using standard poly-A+ selection and rRNA-depletion methods. Averaging the two biologic replicates, poly-A+ selection yielded 327 and 773 more unique lncRNA transcripts for liver and parietal lobe, respectively. More lncRNA were found to be unique to poly-A+ selected libraries, and rRNA-depletion identified small nucleolar RNA (snoRNA) to have a higher relative expression than in the poly-A+ selected libraries. Overall, poly-A+ selection provides a more thorough identification of total lncRNA in equine tissues while rRNA-depletion may allow for easier detection of snoRNAs.

Two main clusters within Trypanosoma cruzi zymodeme 3 are defined by distinct regions of the ribosomal RNA cistron

Parasitology ◽  
2002 ◽  
Vol 124 (2) ◽  
pp. 177-184 ◽  
Author(s):  
M. B. A. MENDONÇA ◽  
N. S. NEHME ◽  
S. S. SANTOS ◽  
E. CUPOLILLO ◽  
N. VARGAS ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Ribosomal Rna ◽  
Southern Blotting ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Pcr Products ◽  
Phylogenetic Lineages ◽  
Definition Of ◽  
Ribosomal Cistron ◽  
Panstrongylus Geniculatus

Trypanosoma cruzi is currently classified into 2 major phylogenetic lineages, T. cruzi I and II, that correlate with the formerly described zymodeme 1 and 2, respectively. Another isoenzymic group (zymodeme 3–Z3) was also described. In this study, we analysed the genetic diversity among Z3 isolates of the Brazilian Amazon by restriction fragment length polymorphism of the intergenic transcribed spacers (ITSs) of the ribosomal RNA cistron and the size of the divergent domain D7 of the 24Sα rRNA gene. DNAs from 12 T. cruzi Z3 isolates obtained from humans (2), Panstrongylus geniculatus (1), and Rhodnius brethesi (9) were submitted to PCR amplification of the ITSs plus the 5·8S rDNA. The PCR products were digested with 4 distinct endonucleases and the profiles analysed by a numerical methodology. The phenetic dendrogram revealed a clear dichotomy in the Z3 group, defining 2 groups that were named Z3-A and Z3-B. Dimorphism was also found in the band sizes of the amplified D7 divergent domain of the 24Sα rDNA, which showed a perfect correlation with the ITSs clustering. The organization of the ribosomal cistron was investigated by Southern blotting and shown to be conserved in the genome of the 2 Z3 groups. This study shows that the rDNA cistron allows the definition of 2 distinct subclusters in Z3 isolates.

scholarly journals The Molecular Basis and Therapeutic Potential of Let-7 MicroRNAs against Colorectal Cancer

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
Rei Mizuno ◽  
Kenji Kawada ◽  
Yoshiharu Sakai
Keyword(s):  
Colorectal Cancer ◽  
Molecular Basis ◽  
Posttranscriptional Regulation ◽  
Therapeutic Agent ◽  
Therapeutic Potential ◽  
Noncoding Rnas ◽  
Intestinal Tumorigenesis ◽  
C Elegans ◽  
Microrna Family ◽  
Underlying Mechanisms

Although a number of studies have revealed the underlying mechanisms which regulate the development of colorectal cancer (CRC), we have not completely overcome this disease yet. Accumulating evidence has shown that the posttranscriptional regulation by the noncoding RNAs such as microRNAs plays an important role in the development or progression of CRC. Among a number of microRNAs, the let-7 microRNA family that was first discovered in C. elegans and conserved from worms to humans has been linked with the development of many types of cancers including CRC. The expression level of let-7 microRNAs is temporally low during the normal developmental processes, while elevated in the differentiated tissues. The let-7 microRNAs regulate the cell proliferation, cell cycle, apoptosis, metabolism, and stemness. In CRC, expressions of let-7 microRNAs have been reported to be reduced, and so let-7 microRNAs are considered to be a tumor suppressor. In this review, we discuss the mechanisms regulating the let-7 microRNA expression and the downstream targets of let-7 in the context of intestinal tumorigenesis. The application of let-7 mimics is also highlighted as a novel therapeutic agent.

scholarly journals Evaluation and utility of mitochondrial ribosomal genes for molecular systematics of parasitic nematodes

2020 ◽  
Author(s):  
Abigail Hui En Chan ◽  
Kittipong Chaisiri ◽  
Serge Morand ◽  
Naowarat Saralamba ◽  
Urusa Thaenkham
Keyword(s):  
Genetic Marker ◽  
Genetic Markers ◽  
Molecular Systematics ◽  
Ribosomal Rna ◽  
Ribosomal Rna Genes ◽  
16S Ribosomal Rna ◽  
12S Rrna ◽  
Rrna Gene ◽  
12S Rrna Gene ◽  
Rna Genes

Abstract Background Molecular advances have accelerated our understanding of nematode systematics and taxonomy. However, comparative analyzes between various genetic markers have led to discrepancies in nematode phylogenies. This study aimed to evaluate the suitability of using mitochondrial 12S and 16S ribosomal RNA genes for nematode molecular systematics. Methods To study the suitability of mitochondrial 12S and 16S ribosomal RNA genes as genetic markers for nematode molecular systematics, we compared them with the other commonly used genetic markers, nuclear internal transcribed spacer 1 and 2 regions, nuclear 18S and 28S ribosomal RNA genes, and mitochondrial cytochrome c oxidase subunit 1 gene. After that, phylum-wide primers for mitochondrial 12S and 16S ribosomal RNA genes were designed, and parasitic nematodes of humans and animals from 75 taxa with 21 representative species were inferred through phylogenetic analyzes. Phylogenetic analyzes were carried out using maximum likelihood and Bayesian inference algorithms. Results The phylogenetic relationships of nematodes based on the mitochondrial 12S rRNA gene supported the monophyly of nematodes in clades I, IV, and V, reinforcing the potential of this gene as a genetic marker for nematode systematics. In contrast, the mitochondrial 16S rRNA gene only supported the monophyly of clades I and V, providing evidence that the 12S rRNA gene is more suitable for nematode molecular systematics. In this study, subclades of clade III containing various nematode families were not monophyletic when the 16S or 12S rRNA gene was used as the genetic marker. This is similar to the phylogenetic relationship revealed by previous studies using whole mitochondrial genomes as genetic markers. Conclusions This study supports the use of the 12S rRNA gene as a genetic marker for studying the molecular systematics of nematodes to understand intra-phyla relationships. Phylum-wide primers for nematodes using mitochondrial ribosomal genes were prepared, which may enhance future studies. Furthermore, sufficient genetic variation in the mitochondrial 12S and 16S rRNA genes between species also allowed for accurate taxonomy to species level, revealing the potential of these two genes as genetic markers for DNA barcoding.

Basonuclin is associated with the ribosomal RNA genes on human keratinocyte mitotic chromosomes

1999 ◽  
Vol 112 (18) ◽  
pp. 3039-3047 ◽  
Author(s):  
H. Tseng ◽  
J.A. Biegel ◽  
R.S. Brown
Keyword(s):  
Ribosomal Rna ◽  
Zinc Fingers ◽  
Hair Follicles ◽  
Ribosomal Rna Genes ◽  
Rrna Genes ◽  
Control Element ◽  
Rrna Gene ◽  
Metaphase Chromosomes ◽  
Acrocentric Chromosomes ◽  
Rna Genes

Basonuclin is a zinc finger protein mainly expressed in keratinocytes of the basal layer of epidermis and the outer root sheath of hair follicles. It is also found in abundance in the germ cells of testis and ovary. In cultured keratinocytes, basonuclin is associated with chromatin in all phases of the cell cycle, including mitosis. By immunocytochemical methods, we demonstrate here that in mitosis basonuclin is associated with the short arms of the acrocentric chromosomes and with other loci on many metaphase chromosomes of human keratinocytes. Using the evolutionarily highly conserved N-terminal pair of zinc fingers in an electrophoresis mobility shift assay, we demonstrate that the DNA target sequences of basonuclin on the acrocentric chromosomes are likely to be within the promoter region of the 45S rRNA gene transcription unit. DNase I footprinting shows that basonuclin zinc fingers interact with the upstream control element of this promoter, which is necessary for the high level of transcription of the rRNA genes. This result suggests that basonuclin may be a tissue-specific transcription factor for the ribosomal RNA genes.

scholarly journals Three redescriptions in Tintinnopsis (Protista: Ciliophora: Tintinnina) from coastal waters of China, with cytology and phylogenetic analyses based on ribosomal RNA genes

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Yang Bai ◽  
Rui Wang ◽  
Wen Song ◽  
Lifang Li ◽  
Luciana F. Santoferrara ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Coastal Waters ◽  
Phylogenetic Analyses ◽  
Rrna Gene ◽  
Middle Portion ◽  
Closely Related Species ◽  
Molecular Information ◽  
Ciliary Tuft ◽  
Rna Genes ◽  
Rrna Sequences

Abstract Background The taxonomy of tintinnine ciliates is vastly unresolved because it has traditionally been based on the lorica (a secreted shell) and it has only recently incorporated cytological and molecular information. Tintinnopsis, the most speciose tintinnine genus, is also the most problematic: it is known to be non-monophyletic, but it cannot be revised until more of its species are studied with modern methods. Results Here, T. hemispiralis Yin, 1956, T. kiaochowensis Yin, 1956, and T. uruguayensis Balech, 1948, from coastal waters of China, were studied. Lorica and cell features were morphometrically investigated in living and protargol-stained specimens, and sequences of three ribosomal RNA (rRNA) loci were phylogenetically analyzed. The three species show a complex ciliary pattern (with ventral, dorsal, and posterior kineties and right, left, and lateral ciliary fields), but differ in lorica morphology, details of the somatic ciliature and rRNA gene sequences. Tintinnopsis hemispiralis is further distinguished by a ciliary tuft (a ribbon of very long cilia originated from the middle portion of the ventral kinety and extending out of the lorica) and multiple macronuclear nodules. Both T. kiaochowensis and T. uruguayensis have two macronuclear nodules, but differ in the number of somatic kineties and the position of the posterior kinety. Two neotypes are fixed for T. hemispiralis and T. kiaochowensis to stabilize the species names objectively, mainly because of the previous unavailability of type materials. By phylogenetic analysis and comparison with closely-related species, we infer that the ciliary tuft and details such as the commencement of the rightmost kinety in the lateral ciliary field are synapomorphies that may help clarify the systematics of Tintinnopsis-like taxa. Conclusion The redescriptions of three poorly known Tintinnopsis species, namely T. hemispiralis, T. kiaochowensis, and T. uruguayensis firstly revealed their ciliary patterns and rRNA sequences. This study expands knowledge and database of tintinnines and helps in identifying potential synapomorphies for future taxonomic rearrangements.

Molecular data provide new insights into the phylogeny of Cladonotinae (Orthoptera: Tetrigoidea) from China with the description of a new genus and species

Zootaxa ◽  
2020 ◽  
Vol 4809 (3) ◽  
pp. 547-559
Author(s):  
RONG-JIAO ZHANG ◽  
CONG-LIN ZHAO ◽  
FEI-PENG WU ◽  
WEI-AN DENG
Keyword(s):  
Ribosomal Rna ◽  
Phylogenetic Relationships ◽  
New Genus ◽  
Molecular Data ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Mitochondrial Cytochrome Oxidase Subunit ◽  
Close Relationship ◽  
Phylogenetic Framework ◽  
Mitochondrial Cytochrome Oxidase

Considerable effort has been devoted to elucidating the phylogenetic relationships of tetrigides. However, there is still no commonly accepted phylogenetic hypothesis. Therefore, the phylogenetic relationships among some subfamilies remain unclear; e.g., Cladonotinae is a controversial group, in which the phylogenetic relationships between genera and the boundaries of some of the included genera are unclear, causing some of the taxa to be difficult to identify. Therefore, an in-depth phylogenetic analysis of Cladonotinae is urgently needed. In this study, a robust phylogenetic framework for the tetrigides was reconstructed based on the combined mitochondrial cytochrome oxidase subunit I (COI), 16S ribosomal RNA (16S rRNA), and nuclear 18S ribosomal RNA (18S rRNA) gene sequences of 25 species belonging to 16 genera of Tetrigoidea from China, which included 13 species from 8 genera of Cladonotinae. Phylogenetic inferences were performed using the combined dataset and Bayesian inference (BI) and Maximum Parsimony (MP) methods, and the phylogenetic tree of Cladonotinae was reconstructed. All inferences based on the results of the present study supported the Cladonotinae subfamily as a polyphyletic group; within the Cladonotinae subfamily, Tetradinodula, and Tuberfemurus were closely related to Tetriginae, while Austrohancockia and Gibbotettix showed a close relationship to the Scelimenidae subfamily. Additionally, a new genus and new species of the Cladonotinae subfamily are described and illustrated: Hainantettix Deng, gen. nov. and Hainantettix strictivertex Deng, sp. nov. 

scholarly journals Prevalence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in dairy cattle in Gansu, northwest China

Parasite ◽  
2020 ◽  
Vol 27 ◽  
pp. 62
Author(s):  
Yilin Wang ◽  
Jianke Cao ◽  
Yankai Chang ◽  
Fuchang Yu ◽  
Sumei Zhang ◽  
...  
Keyword(s):  
Dairy Cattle ◽  
Ribosomal Rna ◽  
Triosephosphate Isomerase ◽  
Giardia Duodenalis ◽  
Northwest China ◽  
Small Subunit ◽  
Gastrointestinal Parasites ◽  
Rrna Gene ◽  
And Control

Cryptosporidium spp. and Giardia duodenalis are common gastrointestinal parasites with a broad range of hosts, including humans, livestock, and wildlife. To examine the infection status and assess the zoonotic potential of Cryptosporidium spp. and G. duodenalis in dairy cattle in Gansu, China, a total of 1414 fecal samples were collected from the rectum, with one sample collected from each individual animal. All the samples were tested using nested PCR based on the small subunit ribosomal RNA (SSU rRNA) gene of Cryptosporidium spp. and G. duodenalis. The overall infection rates of Cryptosporidium spp. and Giardia duodenalis were 4.2% (n = 59) and 1.0% (n = 14), respectively. Four Cryptosporidium species were identified: C. andersoni (n = 42), C. parvum (n = 12), C. bovis (n = 5), and C. ryanae (n = 1). In further analyses of subtypes of C. parvum isolates based on the 60 kDa glycoprotein (gp60) gene, five were successfully subtyped as IIdA19G1 (n = 4) and IIdA15G1 (n = 1). All 14 G. duodenalis isolates were identified as assemblage E using the triosephosphate isomerase (tpi) gene. The relatively low positive rates of Cryptosporidium spp. and G. duodenalis detected here and the predominance of non-human pathogenic species/assemblages of these parasites indicated their unique transmission dynamics in this area and the low level of threat posed to public health. However, continuous monitoring and further studies of these parasites should be conducted for the prevention and control of these pathogens.

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