The stress kit: A new method based on competitive reverse transcriptase-polymerase chain reaction to quantify the expression of human αB-crystallin, Hsp27, and Hsp60
| Autor: | Bajramović, J. J., Geutskens, S. B., Bsibsi, M., Boot, M., Hassankhan, R., Verhulst, K. C., Johannes van Noort |
|---|---|
| Přispěvatelé: | Div. Immunological and Infect. Dis., TNO Prevention and Health, PO Box 2215, 2301 CE Leiden, Netherlands |
| Jazyk: | angličtina |
| Rok vydání: | 2000 |
| Předmět: |
DNA
Complementary heat shock protein 27 complementary DNA Astrocytoma Stress Binding Competitive reverse transcription polymerase chain reaction crystallin Tumor Cells Cultured Humans controlled study RNA Messenger RNA Neoplasm Biology Heat-Shock Proteins messenger RNA Brain Neoplasms Reverse Transcriptase Polymerase Chain Reaction article standard Chaperonin 60 technique heat shock Crystallins Heat beta actin Neoplasm Proteins gene induction priority journal gene expression Reagent Kits Diagnostic heat shock protein 60 |
| Zdroj: | Scopus-Elsevier Cell Stress and Chaperones, 1, 5, 30-35 |
| Popis: | We describe a reverse transcriptase-polymerase chain reaction method for the semiquantitative detection of mRNAs encoding the human heat shock proteins αβ-crystallin, Hsp27, and Hsp60. The method involves the coamplification of cellular mRNA-derived cDNA with a dilution series of a competitor fragment (internal standard), using 1 primer pair common to both templates. Internal standards were based on cellular-derived cDNA engineered to be slightly smaller to differentiate between the target and the standard on electrophoretic separation. Initial cDNA quantitations can be corrected for possible variations during cDNA synthesis by standardizing to the levels of β-actin-encoding cDNA. We show that the coamplified templates accumulate in a parallel manner with the cellular-derived cDNA throughout both the exponential and the nonexponential phase of amplification. Furthermore, we illustrate the utility of this technique by quantifying increased expression of αβ-crystallin, Hsp27, and Hsp60 mRNA in astroglioma cells on heat shock. Chemicals/CAS: Chaperonin 60; Crystallins; DNA, Complementary; Heat-Shock Proteins; HSPB1 protein, human; Neoplasm Proteins; Reagent Kits, Diagnostic; RNA, Messenger; RNA, Neoplasm |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|