Search results for "RNA extraction"
showing 10 items of 30 documents
Routine HCV PCR screening of blood donations to identify early HCV infection in blood donors lacking antibodies to HCV
2001
BACKGROUND: Detection of early hepatitis C infection of blood donors is still a major problem for blood transfusion. Common anti-HCV screening assays show differences in sensitivity and specificity. The often mild symptoms of acute hepatitis C also cause difficulties in the identification of early HCV infection. The feasibility and efficacy of routine screening of blood donations for HCV RNA were investigated. STUDY DESIGN AND METHODS: Blood donations (n = 251,737) were screened for HCV RNA over 4 years. RNA extraction, amplification, and detection were done by two commercial HCV PCR kits (HCV Cobas Amplicor and HCV Cobas Amplicor 2.0, Roche Diagnostics). Screening was done by pool testing …
Cloning and characterization of new orphan nuclear receptors and their developmental profiles duringTenebriometamorphosis
1999
Five PCR fragments corresponding to a part of the DNA-binding domain of different hormone nuclear receptors were isolated from Tenebrio molitor mRNAs. The sequence identity of three of them with known Drosophila nuclear receptors strongly suggests that they are the Tenebrio orthologs of seven-up, DHR3 and β-FTZ-F1, and thus named Tmsvp, TmHR3 and TmFTZ-F1. The full-length sequences of the other two were established. TmHR78 is either a new receptor of the DHR78 family or the same gene which has evolved rapidly, particularly in the E domain. TmGRF belongs to the GCNF1 family and its in vitro translated product binds to the extended half site TCAAGGTCA with high affinity. The periods of expres…
Comparing analytical methods to detect SARS-CoV-2 in wastewater
2021
Wastewater based epidemiology (WBE) has emerged as a reliable strategy to assess the coronavirus disease 2019 (COVID-19) pandemic. Recent publications suggest that SARS-CoV-2 detection in wastewater is technically feasible; however, many different protocols are available and most of the methods applied have not been properly validated. To this end, different procedures to concentrate and extract inactivated SARS-CoV-2 and surrogates were initially evaluated. Urban wastewater seeded with gamma-irradiated SARS-CoV-2, porcine epidemic diarrhea virus (PEDV), and mengovirus (MgV) was used to test the concentration efficiency of an aluminum-based adsorption-precipitation method and a polyethylene…
A method to isolate cDNA-quality RNA from adult conifer needles and a psbA cDNA from Norway spruce
1996
Summary In order to investigate the expression of the psbA gene in damaged and undamaged Norway spruce trees ( Picea abies ) a cDNA clone encoding the D1 protein was isolated via RT-PCR. Applying a method developed by Schneiderbauer et al. (1991) with some modifications, we were able to obtain the required RNA from mature needles and successfully reverse transcribe it into cDNA. Sequence analysis of the cDNA clone revealed an open reading frame (ORF) encoding a 353 amino acid polypeptide that is highly homologous to the D1 protein sequences deduced from higher plant psbA genes. A 4 bp insertion, directly following the stop codon ochre (TAA), was found by comparison with two Pinus species, t…
Selective MicroRNA-Offset RNA Expression in Human Embryonic Stem Cells
2015
Small RNA molecules, including microRNAs (miRNAs), play critical roles in regulating pluri-potency, proliferation and differentiation of embryonic stem cells. miRNA-offset RNAs (moRNAs) are similar in length to miRNAs, align to miRNA precursor (pre-miRNA) loci and are therefore believed to derive from processing of the pre-miRNA hairpin sequence. Recent next generation sequencing (NGS) studies have reported the presence of moRNAs in human neurons and cancer cells and in several tissues in mouse, including pluripotent stem cells. In order to gain additional knowledge about human moRNAs and their putative development-related expression, we applied NGS of small RNAs in human embryonic stem cel…
Diagnosing HIV infection using flow cytometry: From antigenic analyses to a specifically dedicated bead-based assay to measure viral load.
2009
SINCE the discovery of HIV in 1984 (1,2), millions have been infected and have died of AIDS. In 2006, the World Health Organization identified 40 million HIV-infected subjects, noting that antiretroviral therapy was required throughout the world (Fig. 1; http://www.who.int/hiv/data/en/index.html). Today, the situation is very similar and the HIV pandemic remains a major world health problem (3). The treatment of AIDS with highly active antiretroviral therapy (HAART) involves regular monitoring of different blood parameters, requiring the development of convenient and accurate methods that can evaluate HIV infection even in resource-poor countries (4,5). In addition to CD4 cell count, which …
Gene expression of stem cells at different stages of ontological human development.
2013
Abstract Objectives To compare multipotent mesenchymal stem cells (MSCs) obtained from chorionic villi (CV), amniotic fluid (AF) and placenta, with regard to their phenotype and gene expression, in order to understand if MSCs derived from different extra-embryonic tissues, at different stages of human ontological development, present distinct stemness characteristics. Study design MSCs obtained from 30 samples of CV, 30 of AF and 10 placentas (obtained from elective caesarean sections) were compared. MSCs at second confluence cultures were characterized by immunophenotypic analysis with flow cytometry using FACS CANTO II. The expression of the genes Oct-4 (Octamer-binding transcription fact…
Total RNA-isolation of abdominal hernia of rats for quantitative real-time reverse transcription (RT) PCR assays.
2007
Abstract Increasing complications in incisional hernia surgery call for novel treatments. A gene expression analysis of injured tissues displays important parameters for tissue regeneration. Until today, no reliable method has been described for a quantitative gene expression analysis of hernia tissues. In this work, a protocol is described for the isolation of DNA‐free total RNA of incisional hernias for the first time. Moreover, real‐time RT PCR assays for collagen type I and III and TGF‐β1 are demonstrated for relative gene expression analyses. Both methods enable relative gene expression analyses of hernia tissues for the first time.
Optimized RNA extraction and northern hybridization in streptomycetes.
2010
Abstract Northern blot hybridization is a useful tool for analyzing transcript patterns. To get a picture of what really occurs in vivo, it is necessary to use a protocol allowing full protection of the RNA integrity and recovery and unbiased transfer of the entire transcripts population. Many protocols suffer from severe limitations including only partial protection of the RNA integrity and/or loss of small sized molecules. Moreover, some of them do not allow an efficient and even transfer in the entire sizes range. These difficulties become more prominent in streptomycetes, where an initial quick lysis step is difficult to obtain. We present here an optimized northern hybridization protoc…
Removing Batch Effects from Longitudinal Gene Expression - Quantile Normalization Plus ComBat as Best Approach for Microarray Transcriptome Data
2016
International audience; Technical variation plays an important role in microarray-based gene expression studies, and batch effects explain a large proportion of this noise. It is therefore mandatory to eliminate technical variation while maintaining biological variability. Several strategies have been proposed for the removal of batch effects, although they have not been evaluated in large-scale longitudinal gene expression data. In this study, we aimed at identifying a suitable method for batch effect removal in a large study of microarray-based longitudinal gene expression. Monocytic gene expression was measured in 1092 participants of the Gutenberg Health Study at baseline and 5-year fol…