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  • Small tissue samples  (1)
  • apoptosis  (1)
  • myeloma  (1)
Materialart
Erscheinungszeitraum
Schlagwörter
  • 1
    Digitale Medien
    Digitale Medien
    Springer
    Pflügers Archiv 433 (1997), S. 664-668 
    ISSN: 1432-2013
    Schlagwort(e): Key words Molecular biology ; Technique ; Small tissue samples ; RT-PCR ; Molecular assay
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Medizin
    Notizen: Abstract  We describe an adapted version of the Chomczynski and Sacchi [Anal Biochem (1987) 162:156–159] RNA isolation procedure that can be performed in less than 1 h on small (〈15 mg) tissue samples, using commonly available reagents. Our modifications included: (1) one rather than two precipitation steps in the aqueous phase with 99% ethanol and (2) elimination of the 1-h incubation step at –20°C. Our adaptations resulted in RNA yield (μg/mg of tissue) and purity (260/280 nm ratios) comparable to those of the original procedure. Furthermore, the isolated RNA was successfully utilized in reverse transcriptase-polymerase chain reaction assays, suggesting that it was essentially free of carry-over contaminants that could inhibit enzymatic reactions. When tested on tissue sample sizes of 7–12 mg, our adapted procedure allowed the recovery of enough total RNA for use in techniques such as Northern blot analysis. Our modified technique is therefore an inexpensive alternative to commercially available kits when isolating good-quality RNA from very small tissue samples, such as those obtained from needle biopsies.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 2
    Digitale Medien
    Digitale Medien
    Springer
    Cytotechnology 34 (2000), S. 131-139 
    ISSN: 1573-0778
    Schlagwort(e): apoptosis ; bcl-xL ; cell growth ; cell viability ; hybridoma ; myeloma
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie , Medizin , Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
    Notizen: Abstract While the ectopic expression of the anti-apoptoticprotein Bcl-2 has been shown to significantly increaseboth cell viability and antibody production in batchculture, some cell lines are refractory to thesemanipulations. For example, the NS/O and theP3x63Ag8.653 murine myelomas, which express highendogenous levels of the Bcl-2 homologue Bcl-xL, areboth resistant to the anti-apoptotic effect of Bcl-2.This indicates that, in these cells, Bcl-2 and Bcl-xLmay be functionally redundant. In order to define therole which Bcl-xL plays in hybridoma cultures, we usedthe Sp2/0-Ag14 cell line. This murine hybridomaexpresses low levels of Bcl-xL and is highly sensitiveto apoptosis induction by cycloheximide (CHX) and byamino acid depletion. Bcl-xL-transfected Sp2/0-Ag14cells were more resistant than the wild type and theplasmid-containing cells to apoptosis induced by CHXand by glutamine depletion. Moreover, when compared tothe vector-transfected control, Bcl-xL-Sp2/0 cellsexhibited a substantial increase in viability instationary batch culture. Interestingly, Sp2/0-Ag14cells overexpressing Bcl-xL showed a growth behaviourthat was similar to the parent myeloma cell lineP3x63Ag8.653. Our results suggest that Bcl-xLexpression levels are sufficient to account for therelative robustness of some hybridoma cell lines instationary batch cultures.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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