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 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 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 Prospective Comparison of Whole-Blood- and Plasma-Based Hepatitis C Virus RNA Detection Systems: Improved Detection Using Whole Blood as the Source of Viral RNA

1999 ◽  
Vol 37 (3) ◽  
pp. 484-489 ◽  
Author(s):  
Jack T. Stapleton ◽  
Donna Klinzman ◽  
Warren N. Schmidt ◽  
Michael A. Pfaller ◽  
Ping Wu ◽  
...  
Keyword(s):  
Whole Blood ◽  
Hcv Rna ◽  
Plasma Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Rna Detection ◽  
Blood Samples ◽  
Prospective Comparison ◽  
Hcv Antibody ◽  
Whole Blood Samples

We previously demonstrated that whole blood contains significantly more hepatitis C virus (HCV) RNA than plasma. To validate the whole-blood-based HCV RNA detection method, a prospective comparison of HCV RNA detection in whole blood and plasma from 50 patients with chronic liver disease was undertaken. Whole-blood and plasma aliquots were independently tested for HCV RNA by reverse transcriptase (RT) PCR assay, and plasma was tested by the Roche Amplicor assay. HCV RNA was detected in 35 of 50 (70%) whole-blood samples by RT-PCR but in only 26 of 50 (52%) plasma samples tested by the Amplicor assay (P < 0.01). HCV RNA was detected in 85% of HCV antibody-positive patients by the whole-blood method compared with 74% of plasma samples by the Amplicor method. The five HCV antibody-positive subjects who were negative by whole-blood-based RT-PCR assay were all receiving interferon therapy and had normal transaminases at the time of testing. HCV RNA was detected in 38% of HCV antibody-negative subjects by the whole-blood-based RT-PCR assay compared with 6.25% of these patients by the Amplicor assay (P < 0.05). There were nine samples in which HCV RNA was detected in whole blood but the Amplicor test was negative. Eight of the nine RNAs prepared from these whole-blood samples tested positive in the Amplicor assay, thus confirming the specificity of our results. This study demonstrates that whole-blood-based HCV RNA detection is more sensitive than currently available commercial tests and that whole-blood RNA is suitable for use in commercial assays.

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

mRNA levels of CD31, CD144, CD146 and von Willebrand factor do not serve as surrogate markers for circulating endothelial cells

2010 ◽  
Vol 104 (08) ◽  
pp. 318-326 ◽  
Author(s):  
Michiel Strijbos ◽  
Brigitte van Krimpen ◽  
Reno Debets ◽  
Jaco Kraan ◽  
Stefan Sleijfer ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Whole Blood ◽  
Circulating Endothelial Cells ◽  
Surrogate Markers ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Blood Samples ◽  
Whole Blood Samples ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryCirculating endothelial cells (CEC) are considered a promising marker to determine the extent of vascular damage. However, currently available and validated CEC enumeration assays are laborious, time consuming and costly, which limits their clinical utility. Here, we evaluated the feasibility of quantifying mRNA levels of the endothelium-associated markers CD31, CD144, CD146 and von Willebrand factor (vWf) in peripheral blood (PB) of healthy donors, patients, and human umbilical veins by real-time reverse transcriptase polymerase chain reaction (RTPCR) and their use as surrogate markers for CEC. Whole blood samples and CD146+ cell-enriched fractions were assessed for mRNA and protein expression of CD31, CD144, CD146 and vWf by RT-PCR and flow cytometry, respectively. We showed the feasibility to detect endothelial mRNA isolated from HUVEC numbers as low as 10. However, no endothelial mRNA could be measure in whole blood samples, and only low levels of CD31 and CD146 mRNA were detected in suspensions of isolated CEC with numbers up to 4,450 CEC per sample. We conclude that mRNA levels of CD31, CD144, CD146 and vWf in whole blood as detected by real time RT-PCR cannot be used as biomarkers for endstage endothelial cells such as CEC.

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 144. Clinical Validation and Performance of a T-cell Immunosequencing Assay to Identify Past SARS-CoV-2 Infection

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S87-S87
Author(s):  
Sudeb C Dalai ◽  
Jennifer N Dines ◽  
Thomas M Snyder ◽  
Rachel M Gittelman ◽  
Tera Eerkes ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cell ◽  
Whole Blood ◽  
Clinical Performance ◽  
Cross Reactivity ◽  
Rt Pcr ◽  
Care Clinic ◽  
Blood Samples ◽  
Percent Agreement ◽  
Whole Blood Samples

Abstract Background Our understanding of the SARS-CoV-2 immune response has critical gaps that are inadequately addressed with available tools. We report the clinical performance of T-Detect COVID, the first T-cell assay to identify prior SARS-CoV-2 infection using T-cell receptor (TCR) sequencing and repertoire profiling from whole blood samples. Methods The T-Detect COVID assay combines high-throughput immunosequencing of the TCRß gene from blood samples with a statistical classifier demonstrating 99.8% specificity for identifying prior SARS-CoV-2 infection. The assay was employed in several retrospective and prospective cohorts to assess primary and secondary Positive Percent Agreement (PPA) with SARS-CoV-2 RT-PCR (N=205; N=77); primary and secondary Negative Percent Agreement (NPA; N=87; N=79); PPA compared to SARS-CoV-2 serology (N=55); and pathogen cross-reactivity (N=38). The real-world performance of the test was also evaluated in a retrospective review of test ordering (N=69) at a single primary care clinic in Park City, Utah. Results In validation studies, T-Detect COVID demonstrated high PPA (97.1% ≥15 days from diagnosis) in subjects with prior PCR-confirmed SARS-CoV-2 infection; high NPA (~100%) in SARS-CoV-2 negative cases; equivalent or higher PPA with RT-PCR compared to two commercial EUA antibody tests; and no evidence of pathogen cross-reactivity. Review of assay use in a single clinic showed 100% PPA with RT-PCR in individuals with past confirmed SARS-CoV-2 vs. 85.7% for antibody testing, 100% agreement with positive antibody results, and positive results in 2/4 convalescent subjects with seroreversion to a negative antibody. In addition, 12/69 (17.3%) individuals with absent or negative RT-PCR tested positive by T-Detect COVID, nearly all of whom had compatible symptoms and/or exposure. TCR positivity was observed up to 12+ months (median 118 days) from the date of positive RT-PCR. Conclusion A T-cell immunosequencing assay shows high clinical performance for identifying past SARS-CoV-2 infection from whole blood samples. This assay can provide additional insights on the SARS-CoV-2 immune response, with practical implications for clinical management, risk stratification, surveillance, assessing vaccine immunity, and understanding long-term sequelae. Disclosures Sudeb C. Dalai, MD, PhD, Adaptive Biotechnologies (Employee, Shareholder) Jennifer N. Dines, MD, Adaptive Biotechnologies (Employee, Shareholder) Thomas M. Snyder, PhD, Adaptive Biotechnologies (Employee, Shareholder) Rachel M. Gittelman, PhD, Adaptive Biotechnologies (Employee, Shareholder) Tera Eerkes, PhD, Adaptive Biotechnologies (Employee, Shareholder) Pashmi Vaney, PhD, Adaptive Biotechnologies (Employee, Shareholder) Sally Howard, PhD, Adaptive Biotechnologies (Employee, Shareholder) Kipp Akers, PhD, Adaptive Biotechnologies (Employee, Shareholder) Lynell Skewis, PhD, Adaptive Biotechnologies (Employee, Shareholder) Anthony Monteforte, PhD, Adaptive Biotechnologies (Employee, Shareholder) Pamela R. Witte, PhD, Adaptive Biotechnologies (Employee, Shareholder) Cristina Wolf, PhD, Adaptive Biotechnologies (Employee, Shareholder) Hans Nesse, PhD, Adaptive Biotechnologies (Employee, Shareholder) Jia Qadeer, PhD, Adaptive Biotechnologies (Employee, Shareholder) Sarah Duffy, PhD, Adaptive Biotechnologies (Employee, Shareholder) Emily Svejnoha, PhD, Adaptive Biotechnologies (Employee, Shareholder) Caroline Taromino, PhD, Adaptive Biotechnologies (Employee, Shareholder) Ian M. Kaplan, PhD, Adaptive Biotechnologies (Employee, Shareholder) John Alsobrook, MD, Adaptive Biotechnologies (Employee, Shareholder) Thomas Manley, MD, Adaptive Biotechnologies (Employee, Shareholder) Lance Baldo, MD, Adaptive Biotechnologies (Employee, Shareholder, Leadership Interest)

scholarly journals A Streamlined CyTOF Workflow To Facilitate Standardized Multi-Site Immune Profiling of COVID-19 Patients

2020 ◽  
Author(s):  
Daniel Geanon ◽  
Brian Lee ◽  
Geoffrey Kelly ◽  
Diana Handler ◽  
Bhaskar Upadhyaya ◽  
...  
Keyword(s):  
Data Acquisition ◽  
Whole Blood ◽  
Immune Cell ◽  
Technical Expertise ◽  
Sample Collection ◽  
Minimal Processing ◽  
Blood Samples ◽  
Immune Phenotyping ◽  
Whole Blood Samples ◽  
Immune Profiling

AbstractMass cytometry (CyTOF) represents one of the most powerful tools in immune phenotyping, allowing high throughput quantification of over 40 single parameters at single-cell resolution. However, wide deployment of CyTOF-based immune phenotyping studies are limited by complex experimental workflows and the need for specialized CyTOF equipment and technical expertise. Furthermore, differences in cell isolation and enrichment protocols, antibody reagent preparation, sample staining and data acquisition protocols can all introduce technical variation that can potentially confound integrative analyses of large data-sets of samples processed across multiple labs.Here, we describe the validation of a streamlined whole blood CyTOF workflow that addresses many of these challenges and facilitates wider adoption of CyTOF immune monitoring across sites with limited technical expertise or sample-processing resources or equipment. Our workflow utilizes commercially available reagents including the Fluidigm MaxPar Direct Immune Profiling Assay, a dry tube 30-marker immunophenotyping panel, and SmartTube Proteomic Stabilizer, which allows for simple and reliable fixation and cryopreservation of whole blood samples. We validate a workflow that allows for streamlined staining of whole blood samples with minimal processing requirements or expertise at the site of sample collection, followed by shipment to a central CyTOF core facility for batched downstream processing and data acquisition. We further demonstrate the application of this workflow to characterize immune responses in a cohort of hospitalized COVID-19 patients, highlighting key disease-associated changes in immune cell frequency and phenotype.

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.

Sign in / Sign up

Export Citation Format

Share Document