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At least 253 records · Page 14Linked to original sources
PubMed2026

Genome-Wide Impact of Human DBR1 Depletion on RNA Processing Networks Reveal a Connection Between Pre-mRNA Splicing, mRNA Surveillance and Stress Granule Dynamics.

The RNA lariat debranching enzyme DBR1 is essential for intron turnover and RNA metabolism, yet its broader impact on transcriptome regulation remains incompletely defined. To elucidate the consequences of DBR1 depletion, we performed transcriptome-wide RNA sequencing of DBR1-knockdown and wild-type HEK293 cells. Differential expression analysis revealed widespread perturbations in pathways linked to RNA splicing, mRNA surveillance, translational control, and stress-granule biology. Many of the most significantly altered transcripts encode splicing factors and RNA quality-control components, underscoring DBR1's influence on post-transcriptional regulation. Alternative splicing analysis showed changes across multiple event types, with exon skipping accounting for >50% of events, followed by mutually exclusive exons, alternative 5' and 3' splice sites, and retained introns, indicating that DBR1 depletion induces pervasive splicing defects. Direct spliceosome inhibition using isoginkgetin (blocks tri-snRNP recruitment) and pladienolide B (targets SF3B1) reproduced the DBR1-KD mis-splicing patterns of cell signaling genes and factors involved in RNA metabolism, supporting a functional link between DBR1 activity and alternative splicing. Notably, DBR1 knockdown revealed a subset of transcripts that are both NMD-sensitive and enriched within stress granules. Consistent with this observation, G3BP1 immunopurification and confocal microscopy further support a role for DBR1 and UPF1 in stress-granule dynamics, suggesting that these factors may participate at distinct stages to influence mRNA fate under stress conditions. Together, these findings indicate that DBR1 functions beyond lariat RNA turnover as a common regulator of RNA processing, transcriptome stability, and stress granule homeostasis, revealing intricate crosstalk between RNA splicing and RNA quality control pathways in human cells.

Humans↗
PubMed2005

[RNA isolation from human embryonic tissues].

To investigate the method of RNA isolation from human embryonic tissues and the factors influencing the quality of RNA, the RNA from human embryonic tissues obtained with drug-induced labor or non-drug induced labor were isolated by using grind with liquid nitrogen or homogenizer without liquid nitrogen. The results showed that the positive rates of RNA integrity in grind with liquid nitrogen group and in homogenizer without liquid nitrogen group were 68.42% and 29.79% respectively, and there was significant difference between these two groups; however, there was no statistic difference in positive rate of RNA integrity, OD(260)/OD(280) ratio and beta-actin gene expression level between the drug-induced labor group and non-drug induced labor group. It is concluded that pulverize of tissue in liquid nitrogen remains the integrity of RNA isolated and may be applied for RNA isolation from human embryonic tissues. The quality of RNA is not affected by different methods of induction of maternal labor.

Embryo, Mammalian↗
PubMed2024

Heat Inactivation of Nipah Virus for Downstream Single-Cell RNA Sequencing Does Not Interfere with Sample Quality.

Single-cell RNA sequencing (scRNA-seq) technologies are instrumental to improving our understanding of virus-host interactions in cell culture infection studies and complex biological systems because they allow separating the transcriptional signatures of infected versus non-infected bystander cells. A drawback of using biosafety level (BSL) 4 pathogens is that protocols are typically developed without consideration of virus inactivation during the procedure. To ensure complete inactivation of virus-containing samples for downstream analyses, an adaptation of the workflow is needed. Focusing on a commercially available microfluidic partitioning scRNA-seq platform to prepare samples for scRNA-seq, we tested various chemical and physical components of the platform for their ability to inactivate Nipah virus (NiV), a BSL-4 pathogen that belongs to the group of nonsegmented negative-sense RNA viruses. The only step of the standard protocol that led to NiV inactivation was a 5 min incubation at 85 °C. To comply with the more stringent biosafety requirements for BSL-4-derived samples, we included an additional heat step after cDNA synthesis. This step alone was sufficient to inactivate NiV-containing samples, adding to the necessary inactivation redundancy. Importantly, the additional heat step did not affect sample quality or downstream scRNA-seq results.

Nipah Virus↗
PubMed2002

Interactions between fission yeast Cdk9, its cyclin partner Pch1, and mRNA capping enzyme Pct1 suggest an elongation checkpoint for mRNA quality control.

RNA polymerase II (pol II) is subject to an early elongation delay induced by negative factors Spt5/Spt4 and NELF, which is overcome by the positive factor P-TEFb (Cdk9/cyclin T), a protein kinase that phosphorylates the pol II C-terminal domain (CTD) and the transcription elongation factor Spt5. Although the rationale for this arrest and restart is unclear, recent studies suggest a connection to mRNA capping, which is coupled to transcription elongation via physical and functional interactions between the cap-forming enzymes, the CTD-PO(4), and Spt5. Here we identify a novel interaction between fission yeast RNA triphosphatase Pct1, the enzyme that initiates cap formation, and Schizosaccharomyces pombe Cdk9. The C-terminal segment of SpCdk9 comprises a Pct1-binding domain distinct from the N-terminal Cdk domain. We show that the Cdk domain interacts with S. pombe Pch1, a homolog of cyclin T, and that the purified recombinant SpCdk9/Pch1 heterodimer can phosphorylate both the pol II CTD and the C-terminal domain of S. pombe Spt5. We provide genetic evidence that SpCdk9 and Pch1 are functional orthologs of the Saccharomyces cerevisiae CTD kinase Bur1/Bur2, a putative yeast P-TEFb. Mutations of the kinase active site and the regulatory T-loop of SpCdk9 abolish its activity in vivo. Deleting the C-terminal domain of SpCdk9 causes a severe growth defect. We suggest a model whereby Spt5-induced arrest of early elongation ensures a temporal window for recruitment of the capping enzymes, which in turn attract Cdk9 to alleviate the arrest. This elongation checkpoint may avoid wasteful rounds of transcription of uncapped pre-mRNAs.

Amino Acid Sequence↗
PubMed2005

Gene expression profiling of gastric cancer by microarray combined with laser capture microdissection.

AIM: To examine the gene expression profile of gastric cancer (GC) by combination of laser capture microdissection (LCM) and microarray and to correlate the profiling with histological subtypes. METHODS: Using LCM, pure cancer cells were procured from 45 cancerous tissues. After procurement of about 5000 cells, total RNA was extracted and the quality of RNA was determined before further amplification and hybridization. One microgram of amplified RNA was converted to cDNA and hybridized to cDNA microarray. RESULTS: Among 45 cases, only 21 were qualified for their RNAs. A total of 62 arrays were performed. These included 42 arrays for cancer (21 cases with dye-swab duplication) and 20 arrays for non-tumorous cells (10 cases with dye-swab duplication) with universal reference. Analyzed data showed 504 genes were differentially expressed and could distinguish cancerous and non-cancerous groups with more than 99% accuracy. Of the 504 genes, trefoil factors 1, 2, and 3 were in the list and their expression patterns were consistent with previous reports. Immunohistochemical staining of trefoil factor 1 was also consistent with the array data. Analyses of the tumor group with these 504 genes showed that there were 3 subgroups of GC that did not correspond to any current classification system, including Lauren's classification. CONCLUSION: By using LCM, linear amplification of RNA, and cDNA microarray, we have identified a panel of genes that have the power to discriminate between GC and non-cancer groups. The new molecular classification and the identified novel genes in gastric carcinogenesis deserve further investigations to elucidate their clinicopathological significance.

Gene Expression Profiling↗
PubMed2003

Quality control of ribosomal RNA mediated by polynucleotide phosphorylase and RNase R.

Despite their overall accuracy, errors in macromolecular processes, such as rRNA synthesis and ribosome assembly, inevitably occur. However, whether these errors are remediated and how this might be accomplished is not known. In previous work, we showed that a double mutant strain lacking both polynucleotide phosphorylase (PNPase) and RNase R activities is inviable. In the course of examining the molecular basis for this phenotype, we found that shifting a temperature-sensitive mutant strain to 42 degrees C led to cessation of growth and loss of cell viability. Northern analysis of RNA isolated from such cells after the temperature shift revealed that fragments of 16S and 23S rRNA accumulated to a high level, and that the amount of ribosomes and ribosomal subunits decreased due to defects in ribosome assembly. rRNA fragments were not detected at 31 degrees C or when single mutant strains were grown at 42 degrees C. Pulse-chase analysis showed that the rRNA fragments appeared within 5 min at 42 degrees C, and that they accumulated before the loss of cell viability. The data are consistent with a model in which PNPase and RNase R mediate a previously unknown quality control process that normally removes defective rRNAs as soon as they are generated. In the absence of these RNases, rRNA fragments accumulate, leading to interference with ribosome maturation and ultimately to cell death.

Base Sequence↗
PubMed2004

Impact of fixative on recovery of mRNA from paraffin-embedded tissue.

Due to the evolution of advanced tissue-analysis tools, such as proteomics and functional and structural genomics, the demands for handling and preserving samples are changing. For gene expression analysis, the presence of intact and extractable messenger RNA in the test material is mandatory. To find an optimal fixative for tissues aimed for such analyses, we evaluated the morphology-, protein antigen-, and RNA-maintaining abilities of 2 precipitating tissue fixatives, methanol-acetone and Carnoy's. Both fixatives preserved the morphology and protein epitopes of tissues and allowed extraction of total RNA that was of significantly higher quality than RNA extracted from formalin-fixed tissue. Carnoy's fixative performed better than methanol-acetone in maintaining the integrity of RNA, especially when the fixed, paraffin-embedded tissue blocks were stored at room temperature for more than 3 months. Total RNA extracted from epithelial cells microdissected from Carnoy's-fixed tissue samples contained intact template for up to a 977-base pair (bp) amplicon for beta-actin. Because of the emerging role of gene expression analyses in research, and in clinical work in the near future, an RNA-preserving fixative should replace formalin as the primary human tissue fixative. According to our data, Carnoy's fixative is an excellent candidate for a new primary fixing reagent for human tissue samples.

Acetic Acid↗
PubMed2006

Detection of BCR-ABL mutations and resistance to imatinib mesylate.

The major mechanism of imatinib resistance for patients with chronic myeloid leukemia (CML) is clonal expansion of leukemic cells with mutations in the Bcr-Abl fusion tyrosine kinase that reduce the capacity of imatinib to inhibit kinase activity. The early detection of such mutations may allow timely treatment intervention to prevent or overcome resistance. Direct sequencing of the BCR-ABL kinase domain is relatively rapid and allows detection of emerging mutations at a sensitivity of approx 20%. Mutations have been detected over a range of 242 amino acids, which spans the entire kinase domain. For optimal sensitivity, the kinase domain of the abnormal gene should be isolated by reverse-transcription (RT) polymerase chain reaction (PCR) amplification using primers that hybridize to the BCR and ABL genes. The quality of the RNA is assessed by real-time quantitative PCR prior to analysis, and BCR-ABL levels are determined. Only RNA of adequate quality is used to ensure accurate and reproducible mutation analysis. Depending on the level of BCR-ABL transcripts, a one- or two-step PCR is required to amplify the kinase domain. Direct sequencing with dye terminator chemistry is performed using PCR-purified products. The sequence is compared to an ABL kinase domain reference sequence using sequencing analysis software, which aligns the sequences and highlights single or multiple mutations.

Amino Acid Substitution↗
PubMed2003

Computational methods and evaluation of RNA stabilization reagents for genome-wide expression studies.

Gene expression studies require high quality messenger RNA (mRNA) in addition to other factors such as efficient primers and labeling reagents. To prevent RNA degradation and to improve the quality of gene array expression data, several commercial reagents have become available. We examined a conventional hot-phenol lysis method and RNA stabilization reagents, and generated comparative gene expression profiles from Escherichia coli cells grown on minimal medium. Our data indicate that certain RNA stabilization reagents induce stress responses and proper caution must be exercised during their use. We observed that the laboratory reagent (phenol/EtOH, 5:95, v/v) worked efficiently in isolating high quality mRNA and reproducibility was such that reliable gene expression profiles were generated. To assist in the analysis of gene expression data, we wrote a number of macros that use the most recent gene annotation and process data in accordance with gene function. Scripts were also written to examine the occurrence of artifacts, based on GC content, length of the individual open reading frame (ORF), its distribution on plus and minus DNA strands, and the distance from the replication origin.

Base Composition↗
PubMed2006

Cervical tissue collection methods for RNA preservation: comparison of snap-frozen, ethanol-fixed, and RNAlater-fixation.

Promising molecular techniques may allow for testing of novel and complex hypotheses such as defining gene expression profiles in specific cells, tumors, or their microenvironments. For most large cancer epidemiologic and population-based studies, however, application of such promising techniques may not be possible owing to constraints of specimen preservation from paraffin-embedded tissues. Alternative methods would ideally preserve tissue morphology and not degrade DNA or RNA. We conducted a comparison of snap-freezing (freezing with liquid nitrogen), ethanol-fixation with low melt polyester wax embedding, and RNAlater-preservation techniques to determine which method was optimal for subsequent assessment of gene expression changes in cervical cancer. From each of 15 women with cancer and 30 without, we procured 3 pieces of cervical tissue and compared snap-freezing, ethanol-fixation, and RNAlater-preservation techniques. Despite slight loss in morphologic quality from snap-frozen cervix tissues, RNA quality was equivalent to or better than RNAlater-preserved tissues and significantly exceeded that from ethanol-fixed/polyester wax embedded tissue. In conclusion, despite the moderate logistical constraints in set-up that required either liquid nitrogen or dry ice on-site for snap-freezing tissue, the ease of downstream processing and consistent high quality RNA made it preferable to the other 2 methods.

Cervix Uteri↗
PubMed2003

Improved method for generating microarray probes using submicrogram amounts of total RNA.

The ability to identify disease-associated genes using microarray technology is dependent on isolating high-quality total RNA from the diseased tissue under study. However, it is not always possible to obtain large amounts of affected tissue from patients and therefore the quality and yield of RNA may be compromised. This protocol described a procedure for generating superior microarray probes by amplifying RNA sequences via successive rounds of in vitro transcription (IVT) reactions. This improved procedure utilizes a 9-mer primer to generate the IVT template, which is able to recapitulate the size ditribution of the original isolated RNA sample.

Algorithms↗
PubMed1996

Analysis of tumor spillage during radical prostatectomy using RT-PCR of prostate specific antigen.

Recent reports have suggested the shedding of cancer cells during radical extirpation of tumors. Prostate cells can be expressed from the prostate ex vivo and found in the expressed prostatic secretions. We conducted an in vivo study to determine if prostate epithelial cells can be found in the operative site as determined by RT-PCR targeted at prostate specific antigen (PSA) and to correlate this with pathologic stage and outcome. We analyzed 14 consecutive radical retropubic prostatectomy procedures with a minimum 1-year follow-up. Intraoperatively, 5-10 ml of fluid (representing blood, urine, and irrigant) was aspirated from the operative field at three time points: after transaction of the dorsal vein complex, urethra, and bladder neck. Ficoll gradient fractionation was carried out on the specimens, and RNA was extracted from the cell pellet. Quality of RNA and presence of the PSA mRNA was determined by RT-PCR targeted at actin and PSA, respectively, using previously published primers. The medical records were reviewed for pathologic stage. There were nine patients with extraprostatic disease and five patients with organ confined disease. Five of 14 (36%) patients tested positive for prostate epithelial cells in the operative field at one or more points during radical prostatectomy. All five positive RT-PCR PSA assays were in patients with locally advanced disease, whereas all of the patients with organ-confined disease were negative for RT-PCR. This preliminary in vivo study suggests that locally advanced prostate cancer may be associated with PSA expressing cells in the operative field during radical prostatectomy. The clinical significance of this is unclear, but this finding suggests that shedding of cells in the operative field is more likely with locally advanced disease.

Combined Modality Therapy↗
PubMed2002

Increased yield of total RNA from fine-needle aspirates for use in expression microarray analysis.

Fine-needle aspirate samples hold the potential for gaining valuable insight into the molecular details and prognostic indicators for certain types of cancer in a limited volume of relatively pure tumor cells. Although limited, such clinical samples can be used with high efficiency when analyzed in conjunction with gene-dense expression microarrays. For this reason, it is essential to retrieve as much high-quality genetic material as possible from each fine-needle aspirate sample. We have conducted a study to improve the efficiency of extracting high quality total RNA to use in microarray analysis from single ex vivo fine-needle aspirate samples of 11 breast cancers added to RNAlater RNA Stabilization Reagent immediately upon collection. Approximately half the total RNA from fine-needle aspirate samples of breast cancers was isolated from the supernatant, and that RNA had similar quality and gene expression profile to the RNA that was isolated from the corresponding cell pellet. We recommend that the supernatant not be discarded when extracting RNA from fine-needle aspirate samples stored in RNAlater.

Biopsy, Needle↗
PubMed2004

RNA integrity as a quality indicator during the first steps of RNP purifications : a comparison of yeast lysis methods.

BACKGROUND: The completion of several genome-sequencing projects has increased our need to assign functions to newly identified genes. The presence of a specific protein domain has been used as the determinant for suggesting a function for these new genes. In the case of proteins that are predicted to interact with mRNA, most RNAs bound by these proteins are still unknown. In yeast, several protocols for the identification of protein-protein interactions in high-throughput analyses have been developed during the last years leading to an increased understanding of cellular proteomics. If any of these protocols or similar approaches shall be used for the identification of mRNA-protein complexes, the integrity of mRNA is a critical factor. RESULTS: We compared the effect of different lysis protocols on RNA integrity. We report dramatic differences in RNA stability depending on the method used for yeast cell lysis. Glass bead milling and French Press lead to degraded mRNAs even in the presence of RNase inhibitors. Thus, they are not suitable to purify intact mRNP complexes or to identify specific mRNAs bound to proteins. CONCLUSION: We suggest a novel protocol, grinding deep-frozen cells, for the preparation of protein extracts that contain intact RNAs, as lysis method for the purification of mRNA-protein complexes from yeast cells.

Cell Fractionation↗
PubMed2002

Postmortem stability of RNA isolated from bovine reproductive tissues.

Molecular biology is being increasingly used to address the complex problem of bovine infertility. One common concern shared by many of these studies is the postmortem delay in obtaining reproductive tissues and the effect this may have on RNA dependent studies. To address this concern, bovine ovarian, oviduct and uterine tissue samples, collected over intervals ranging from 0 to 96 h postmortem to freeze storage, were analysed to determine the potential effects on RNA quantity and quality. The analysis showed that total RNA yields were not changed significantly by postmortem interval up to 96 h while 28S ribosomal RNA remained intact up to 24 h postmortem. Specific messenger RNA transcripts encoding beta-actin, GAPDH and transforming growth factor-beta were detected in all tissues up to 96 h postmortem using reverse transcriptase-polymerase chain reaction and Northern analysis indicated no detectable mRNA degradation up to 24 h postmortem. Finally, using poly(A)(+) mRNA isolated from ovarian tissues frozen 2 h postmortem, we constructed corpus luteum and ovarian cortex cDNA libraries containing 7.65x10(4) and 1.9x10(6) primary transformants with average cDNA lengths of 2.3 and 1.6 kb respectively. Taken together, these data show that a postmortem delay of up to 24 h does not significantly affect the yield or quality of RNA prepared from bovine reproductive tissues.

Actins↗
PubMed2004

Evaluation of quality-control criteria for microarray gene expression analysis.

BACKGROUND: Development of quality-control criteria to ensure reproducibility of microarray results for potential clinical application is still in its infancy. METHODS: In the present studies we developed quality-control criteria and evaluated their effect in microarray data analysis using total RNA from cell lines, frozen tumors, and a commercially available reference RNA. Quality-control criteria such as A(260)/A(280) ratios, percentage of rRNA, and median size of cDNA and cRNA synthesis products were evaluated for robustness in microarray analysis. Furthermore, precision studies using a reference material were performed on the Affymetrix HG-U133A high-density oligonucleotide microarrays. The same reference RNA sample was examined in 16 different chips run on 2 different days in the four different modules of the Affymetrix fluidics workstation. Fresh and frozen fragmented cRNAs were also compared. An ANOVA model was fit to identify the main sources of variation. RESULTS: Good-quality samples showed >30% rRNA in the electropherograms and cDNA and cRNA synthesis products with median sizes of 2.0 and 3.0 kb, respectively. Precision studies showed that the main source of variation was the day-to-day variability, minimally affecting hybridization exogenous control genes. Altogether, the results showed that the Affymetrix Genechip system is highly reproducible when RNA that meet the quality-control criteria are used (overall P >0.01). CONCLUSIONS: These results confirm the need to establish defined quality-control criteria for sample quality to distinguish between analytical and biological variability.

Analysis of Variance↗
PubMed1995

An improved method for isolating RNA from coccidian oocysts.

A method, based on the disruption of eimerian oocysts in a French pressure cell in the presence of guanidine isothiocyanate, has been developed to isolate large quantities of high quality total RNA efficiently. This procedure results in a 12.5-fold greater number of oocysts broken, and a 22-fold greater yield of total RNA than from disruption by conventional grinding in liquid N2. In addition, the RNA isolated by the French pressure cell method was of equal or superior quality when compared to RNA isolated by grinding. This procedure provides a significant improvement in RNA extraction from eimerian oocysts and will greatly facilitate the study of gene expression in this important group of parasites.

Animals↗
PubMed2003

Comparative evaluation of four large-volume RNA extraction kits in the isolation of viral RNA from water samples.

The quality of the RNA extraction system plays a crucial role for the detection of viruses in water or environmental samples. In the present study we investigated the detection limit, the efficiency and the presence of eventually co-extracted inhibitors by comparing four commercially available large scale (>or=1 ml) viral RNA extraction methods (QIAamp Viral RNA Mini Kit in combination with preconcentration by Centricon YM-100 [Centricon-QIAamp], QIAamp UltraSens Virus Kit, NucliSens Isolation Kit and NucleoSpin RNA Virus F). A 1 ml 50 mM glycine (pH 8.0) containing 1% beef extract was spiked with different concentrations of poliovirus vaccine strains, extracted by the four methods and analysed by RT-nested PCR or RT-quantitative LightCycler PCR. Eight replicates were analysed for each concentration on different days. The positive cut-off point was determined to be at 0.25 CCID(50) per ml (Centricon-QIAamp), 1.46 CCID(50) per ml (UltraSens), 0.4 CCID(50) per ml (NucliSens) and 3.03 CCID(50) per ml (NucleoSpin). Quantitative analysis (LightCycler) of a high-titer sample showed significant differences between the efficiencies of the four extraction methods examined. The efficiencies of the extraction methods were normalized to the NucliSens method as follows: (71% Centricon-QIAamp, 18% UltraSens, 100% NucliSens and 23% NucleoSpin). In addition, spiked negative controls did show significant differences, indicating a co-extraction of inhibitors. Compared with the non-inhibited positive control the inhibitions were 21, 37, 27 and 68% for the Centricon-QIAamp, UltraSens, NucliSens and NucleoSpin methods, respectively. Taken together, these findings indicate that of the four evaluated extraction methods both the NucliSens and Centricon-QIAamp are best suited to extract viral RNA from water samples previously concentrated and have shown to be very sensitive, efficient and robust methods.

Base Sequence↗