The Experts below are selected from a list of 306 Experts worldwide ranked by ideXlab platform

Nicoletta Sacchi - One of the best experts on this subject based on the ideXlab platform.

Piotr Chomczynski - One of the best experts on this subject based on the ideXlab platform.

Lalji Singh - One of the best experts on this subject based on the ideXlab platform.

  • Isolation of high-molecular-weight DNA from small samples of blood having nucleated erythrocytes, collected, transported, and stored at room temperature
    Genetic Analysis: Biomolecular Engineering, 1992
    Co-Authors: Ramesh K. Aggarwal, Jeffrey W. Lang, Lalji Singh
    Abstract:

    Blood samples collected in the field for isolating DNA suitable for molecular analysis need special care in their storage and handling. In this article, we describe a simple method for the isolation of good-quality high-molecular-weight DNA that does not require low temperature conditions during collection, storage, and/or transportation of blood samples. This method involves smearing small aliquots of blood onto clean slides and air drying them at room temperature. The slides with blood smears can then be transported or stored at room temperature and still serve as a very good source of high-molecular-weight DNA. Genomic DNA from these samples can be extracted by organic phase separation (Phenol-Chloroform Extraction) after lysis. The DNA thus obtained is of high quality and yields DNA fingerprints qualitatively similar to those prepared from corresponding control DNA isolated from frozen blood samples. Needing minimal facilities at field sites, the method is very convenient for conducting RFLP analysis of wild/field populations for demographic, behavioral, and ecologic studies. © 1992.

E. Schütz - One of the best experts on this subject based on the ideXlab platform.

  • Suitability of ultrasound-guided fine-needle aspiration biopsy for transcriptome sequencing of the canine prostate
    Scientific Reports, 2019
    Co-Authors: H. Thiemeyer, L. Taher, J. T. Schille, L. Harder, S. O. Hungerbuehler, R. Mischke, Z. Kiełbowicz, B. Brenig, Marion Hewicker-trautwein, E. Schütz
    Abstract:

    Ultrasound-guided fine-needle aspiration (US-FNA) biopsy is a widely used minimally invasive sampling procedure for cytological diagnosis. This study investigates the feasibility of using US-FNA samples for both cytological diagnosis and whole transcriptome RNA-sequencing analysis (RNA-Seq), with the ultimate aim of improving canine prostate cancer management. The feasibility of the US-FNA procedure was evaluated intra vitam on 43 dogs. Additionally, aspirates from 31 euthanised dogs were collected for standardising the procedure. Each aspirate was separated into two subsamples: for cytology and RNA Extraction. Additional prostate tissue samples served as control for RNA quantity and quality evaluation, and differential expression analysis. The US-FNA sampling procedure was feasible in 95% of dogs. RNA isolation of US-FNA samples was successfully performed using Phenol-Chloroform Extraction. The extracted RNA of 56% of a subset of US-FNA samples met the quality requirements for RNA-Seq. Expression analysis revealed that only 153 genes were exclusively differentially expressed between non-malignant US-FNAs and tissues. Moreover, only 36 differentially expressed genes were associated with the US-FNA sampling technique and unrelated to the diagnosis. Furthermore, the gene expression profiles clearly distinguished between non-malignant and malignant samples. This proves US-FNA to be useful for molecular profiling.

A. H.j. Kolk - One of the best experts on this subject based on the ideXlab platform.

  • A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples
    J Clin Microbiol, 1994
    Co-Authors: L F Kox, D Rhienthong, A M Miranda, N Udomsantisuk, S Van Heusden, S Kuijper, K. Ellis, J Van Leeuwen, A. H.j. Kolk
    Abstract:

    Diagnostic techniques based on PCR have two major problems: false-positive reactions due to contamination with DNA fragments from previous PCRs (amplicons) and false-negative reactions caused by inhibitors that interfere with the PCR. We have improved our previously reported PCR based on the amplification of a fragment of the Mycobacterium tuberculosis complex-specific insertion element IS6110 with respect to both problems. False-positive reactions caused by amplicon contamination were prevented by the use of uracil-N-glycosylase and dUTP instead of dTTP. We selected a new set of primers outside the region spanned by the formerly used primers to avoid false-positive reactions caused by dTTP-containing amplicons still present in the laboratory. With this new primer set, 16 copies of the IS6110 insertion element, the equivalent of two bacteria, could be amplified 10(10) times in 40 cycles, resulting in a mean efficiency of 77% per cycle. To detect the presence of inhibitors of the Taq polymerase, which may cause false-negative reactions, part of each sample was spiked with M. tuberculosis DNA. The DNA purification method using guanidinium thiocyanate and diatoms effectively removed most or all inhibitors of the PCR. However, this was not suitable for blood samples, for which we developed a proteinase K treatment followed by Phenol-Chloroform Extraction. This method permitted detection of 20 M. tuberculosis bacteria per ml of whole blood. Various laboratory procedures were introduced to reduce failure or inhibition of PCR and avoid DNA cross contamination. We have tested 218 different clinical specimens obtained from patients suspected of having tuberculosis. The samples included sputum (n=145), tissue biopsy samples (n=25), cerebrospinal fluid (n=15), blood (n=14), pleural fluid (n=9), feces, (n=7), fluid from fistulae (n=2), and pus from a wound (n=1). The results obtained by PCR were consistent with those obtained with culture, which is the "gold standard." We demonstrate that PCR is a useful technique for the rapid diagnosis of tuberculosis at various sites.