scholarly journals rRNA Stability in Heat-Killed and UV-Irradiated EnterotoxigenicStaphylococcus aureus and Escherichia coliO157:H7†

1998 ◽  
Vol 64 (11) ◽  
pp. 4264-4268 ◽  
Author(s):  
John L. McKillip ◽  
Lee-Ann Jaykus ◽  
Maryanne Drake
Keyword(s):  
Uv Irradiation ◽  
Foodborne Pathogens ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Brain Heart Infusion ◽  
Northern Blotting ◽  
Extreme Heat ◽  
Heat Inactivation ◽  
Rt Pcr

ABSTRACT Differentiation of viable cells from nonviable cells is of considerable importance in the development of methods to detect foodborne pathogens. To study the suitability of 16S rRNA as an indicator of cell viability in nucleic acid-based detection assays, we examined rRNA stability in two representative foodborne pathogens,Escherichia coli O157:H7 and enterotoxigenicStaphylococcus aureus, which were inactivated by extreme heat, moderate heat, and UV irradiation. Cell death under all conditions was confirmed by a failure to grow in brain heart infusion broth after incubation for 48 h at 37°C. rRNA stability was monitored by a Northern blot analysis, and detection was evaluated by using reverse transcription (RT)-PCR performed with two primer sets (which produced 325- and 1,400-bp amplicons). rRNA of neither pathogen was detected by Northern blot analysis and RT-PCR after cells were killed by autoclaving at 121°C for 15 min. In contrast, intact rRNA of both pathogens were detected by Northern blotting and could be amplified by RT-PCR up to 48 h after cells were killed by heat treatment at 80°C and UV irradiation at 254 nm. rRNA was a suitable target molecule for monitoring bacterial viability under extreme heat conditions, but the presence of rRNA was not correlated with viability following moderate heat inactivation or UV irradiation of cells.

scholarly journals RT-PCR and Northern blot analysis in search for a putative Paramecium beta-adrenergic receptor.

1999 ◽  
Vol 46 (3) ◽  
pp. 813-821
Author(s):  
A Płatek ◽  
J Wiejak ◽  
E Wyroba
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Adrenergic Receptor ◽  
Northern Blot Analysis ◽  
Molecular Probes ◽  
Northern Hybridization ◽  
Rt Pcr ◽  
Beta Adrenergic Receptor ◽  
Beta Adrenergic ◽  
Sequence Tagged Sites

RT-PCR and Northern blot analysis were performed in order to search for a putative beta-adrenergic receptor (beta-AR) in Paramecium using several beta2-adrenergic-specific molecular probes. Under strictly defined RT-PCR conditions DNA species of expected molecular size about 360 bp were generated with the primers corresponding to the universal mammalian beta2-AR sequence tagged sites (located within the 4th and the 6th transmembrane regions of the receptor). This RT-PCR product hybridized in Southern blot analysis with the oligonucleotide probe designed to the highly conservative beta2-AR region involved in G-proteins interaction and located within the amplified region. Northern hybridization was performed on Paramecium total RNA and mRNA with human beta2-AR cDNA and two oligonucleotide probes: the first included Phe 290 involved in agonist binding (Strader et al., 1995) and the second was the backward RT-PCR primer. All these probes revealed the presence of about 2 kb mRNA which is consistent with the size of beta2-AR transcripts found in higher eukaryotes.

scholarly journals Absence of c-kit receptor and absent proliferative response to stem cell factor in childhood Burkitt's lymphoma cells

Blood ◽  
1995 ◽  
Vol 86 (4) ◽  
pp. 1469-1480 ◽  
Author(s):  
J Tomeczkowski ◽  
A Beilken ◽  
D Frick ◽  
B Wieland ◽  
A Konig ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Northern Blot ◽  
Blot Analysis ◽  
Stem Cell Factor ◽  
Northern Blot Analysis ◽  
Thymidine Incorporation ◽  
Rt Pcr ◽  
Low Level

The cytokine stem cell factor (SCF) synergizes with interleukin-7 (IL- 7) to enhance the proliferation of pre-B cells. To examine the role of SCF and its receptor, c-kit, in the pathogenesis of pediatric Burkitt's lymphomas (BL), we investigated the expression of SCF and c-kit in BL cells and the mitogenic activity of SCF on BL cells. A panel of 13 BL cell lines and 7 fresh biopsy tumors was investigated. BL cells were stimulated either by Epstein-Barr virus (EBV) infection or by different reagents and cytokines, and expression of SCF and c-kit was studied on the mRNA level by Northern blot analysis and reverse-transcriptase polymerase chain reaction (RT-PCR), followed by Southern blotting. c- kit expression was also studied by fluorescence-activated cell sorting and by crosslinking of digoxigenin-labeled recombinant human SCF to the cell surface. Proliferation of BL cell lines was measured by 3H- thymidine incorporation. Low-level expression of c-kit mRNA was detected in 2 of 13 unstimulated BL cell lines and in 1 fresh BL tumor. One cell line showed upregulation of c-kit mRNA with A23187 and downregulation with phorbol myristate acetate. Neither c-kit nor SCF could be detected in any other cell line under any condition of stimulation as analyzed by Northern blot analysis, RT-PCR followed by Southern blot analysis, crosslinking, and immunofluorescence. No response to SCF was seen in 3H-thymidine incorporation assays. We conclude that most BL cells express neither SCF nor c-kit and that the low-level expression of c-kit in some BL cells most likely has no biologic significance.

A comparison of RT-PCR and Northern blot analysis in quantifying metallothionein mRNA levels in killifish exposed to waterborne cadmium

1995 ◽  
Vol 39 (1-4) ◽  
pp. 137-141 ◽  
Author(s):  
Lisa Ann Elizabeth Kaplan ◽  
Kathleen Van Cleef ◽  
Isaac Wirgin ◽  
Joseph F. Crivello
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Waterborne Cadmium

scholarly journals Stabilization of mRNA Expression in Whole Blood Samples

2002 ◽  
Vol 48 (11) ◽  
pp. 1883-1890 ◽  
Author(s):  
Lynne Rainen ◽  
Uwe Oelmueller ◽  
Stewart Jurgensen ◽  
Ralf Wyrich ◽  
Cynthia Ballas ◽  
...  
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Whole Blood ◽  
Northern Blot Analysis ◽  
Gene Induction ◽  
Sample Collection ◽  
Rt Pcr ◽  
Blood Samples ◽  
Whole Blood Samples ◽  
Specific Mrna

Abstract Background: Accurate quantification of mRNA in whole blood is made difficult by the simultaneous degradation of gene transcripts and unintended gene induction caused by sample handling or uncontrolled activation of coagulation. This study was designed to compare a new blood collection tube (PAXgeneTM Blood RNA System) and a companion sample preparation reagent set with a traditional sample collection and preparation method for the purpose of gene expression analysis. Methods: We collected parallel blood samples from healthy donors into the new sample collection tubes and control EDTA tubes and performed serial RNA extractions on samples stored for 5 days at room temperature and for up to 90 days at 4 and 20 °C. Samples were analyzed by Northern blot analysis or reverse transcription-PCR (RT-PCR). Results: Specific mRNA concentrations in blood stored in EDTA tubes at any temperature changed substantially, as determined by high-precision RT-PCR. These changes were eliminated or markedly reduced when whole blood was stored in PAXgene tubes. Loss of specific mRNAs, as measured by RT-PCR, reflected total RNA depletion as well as specific mRNA destruction demonstrated by Northern blot analysis. The salutary effects of PAXgene on mRNA stabilization extended to blood samples from eight unrelated donors. Conclusions: Compared with whole blood collected in EDTA tubes and extracted by an organic method, the PAXgene Blood RNA System reduced RNA degradation and inhibited or eliminated gene induction in phlebotomy whole blood samples. Storage of whole blood samples in PAXgene tubes can be recommended for clinically related blood samples that will be analyzed for total or specific RNA content.

scholarly journals Identification and initial characterization of stathmin by the differential display method in nerve growth factor-treated PC12 cells

1998 ◽  
pp. 707-712 ◽  
Author(s):  
K Takekoshi ◽  
F Nomura ◽  
K Isobe ◽  
M Motooka ◽  
T Nammoku ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Pc12 Cells ◽  
Differential Display ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Differentially Expressed ◽  
Rt Pcr ◽  
Differential Display Method

The differential display of mRNA is a new strategy to identify genes that are differentially expressed under altered conditions. We applied this method to determine differential gene expression in the rat pheochromocytoma cell line during differentiation induced by nerve growth factor (NGF). Three different mRNA species were isolated, and their differential expression was confirmed by RT-PCR. One of the mRNA species was identified as stathmin, a 19 kDa cytosolic protein attracting increasing interest for its role in signal transduction. In the NGF-treated PC12 cells, the expression of stathmin mRNA increased in a time-dependent manner, as assessed by northern blot analysis and RT-PCR. We also assessed by northern blot analysis how the expression of stathmin mRNA was altered in human pheochromocytomas (n = 5) compared with that in normal adrenal medulla tissue (n = 5). The mRNA concentrations were found to be significantly greater in the pheochromocytomas than in the normal tissues. It has been shown that stathmin mRNA concentrations are increased in various tumor cells. As pheochromocytomas are well-differentiated tumors of neural origin, it is not unexpected that stathmin mRNA is overexpressed in these tumors. Stathmin was isolated and identified as a differentially expressed gene by the differential display method in PC12 cells during differentiation induced by NGF. In addition, stathmin mRNA was found to be overexpressed in human pheochromocytomas. The mechanisms responsible for the up-regulation of stathmin mRNA during differentiation of PC12 cells and the significance of its overexpression in human pheochromocytomas remain to be determined.

scholarly journals Conservation of the structure and organization of lupin mitochondrial nad3 and rps12 genes.

1998 ◽  
Vol 45 (3) ◽  
pp. 695-699 ◽  
Author(s):  
M Rurek ◽  
M Oczkowski ◽  
H Augustyniak
Keyword(s):  
Nucleotide Sequence ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Lupinus Albus ◽  
Sequence Conservation ◽  
Rt Pcr ◽  
High Level

A high level of the nucleotide sequence conservation of mitochondrial nad3 and rps12 genes was found in four lupin species. The only differences concern three nucleotides in the Lupinus albus rps12 gene and three nucleotides insertion in the L. mutabilis spacer. Northern blot analysis as well as RT-PCR confirmed cotranscription of the L. luteus genes because the transcripts detected were long enough.

scholarly journals SarT, a Repressor of α-Hemolysin inStaphylococcus aureus

2001 ◽  
Vol 69 (8) ◽  
pp. 4749-4758 ◽  
Author(s):  
Katherine A. Schmidt ◽  
Adhar C. Manna ◽  
Steven Gill ◽  
Ambrose L. Cheung
Keyword(s):  
Staphylococcus Aureus ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Basic Protein ◽  
Molecular Size ◽  
Northern Blotting ◽  
Minimal Effect ◽  
Rabbit Erythrocyte ◽  
Hemolytic Assay

ABSTRACT In searching the Staphylococcus aureus genome, we found several homologs to SarA. One of these genes, sarT, codes for a basic protein with 118 residues and a predicted molecular size of 16,096 Da. Northern blot analysis revealed that the expression ofsarT was repressed by sarA and agr. An insertion sarT mutant generated in S. aureusRN6390 and 8325-4 backgrounds revealed minimal effect on the expression of sarR and sarA. The RNAIII level was notably increased in the sarT mutant, particularly in postexponential-phase cells, while the augmentative effect on RNAII was less. SarT repressed the expression of α-hemolysin, as determined by Northern blotting, Western blotting, and a rabbit erythrocyte hemolytic assay. This repression was relieved upon complementation. Similar toagr and sarA mutants, which predictably displayed a reduction in hla expression, the agr sarT mutant exhibited a lower level of hlatranscription than the sarT mutant. In contrast,hla transcription was enhanced in the sarA sarTmutant compared with the single sarA mutant. Collectively, these results indicated that the sarA locus, contrary to the regulatory action of agr, induced α-hemolysin production by repressing sarT, a repressor ofhla transcription.

scholarly journals IL-8 and MCP Gene Expression and Production by LPS-Stimulated Human Corneal Stromal Cells

2012 ◽  
Vol 2012 ◽  
pp. 1-4 ◽  
Author(s):  
Roni M. Shtein ◽  
Susan G. Elner ◽  
Zong-Mei Bian ◽  
Victor M. Elner
Keyword(s):  
Gene Expression ◽  
Northern Blot ◽  
Stromal Cells ◽  
Blot Analysis ◽  
Time Course ◽  
Northern Blot Analysis ◽  
Statistical Significance ◽  
Dependent Manner ◽  
Corneal Inflammation ◽  
Chemotactic Protein

Purpose. To determine time course of effect of lipopolysaccharide (LPS) on production of interleukin-8 (IL-8) and monocyte chemotactic protein (MCP) by cultured human corneal stromal cells.Methods. Human corneal stromal cells were harvested from donor corneal specimens, and fourth to sixth passaged cells were used. Cell cultures were stimulated with LPS for 2, 4, 8, and 24 hours. Northern blot analysis of IL-8 and MCP gene expression and ELISA for IL-8 and MCP secretion were performed. ELISA results were analyzed for statistical significance using two-tailed Student'st-test.Results. Northern blot analysis demonstrated significantly increased IL-8 and MCP gene expression after 4 and 8 hours of exposure to LPS. ELISA for secreted IL-8 and MCP demonstrated statistically significant increases (P<0.05) after corneal stromal cell stimulation with LPS.Conclusions. This paper suggests that human corneal stromal cells may participate in corneal inflammation by secreting potent leukocyte chemotactic and activating proteins in a time-dependent manner when exposed to LPS.

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