Print Email Facebook Twitter 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 Title 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 Author Bajramović, J.J. Geutskens, S.B. Bsibsi, M. Boot, M. Hassankhan, R. Verhulst, K.C. van Noort, J.M. Div. Immunological and Infect. Dis., TNO Prevention and Health, PO Box 2215, 2301 CE Leiden, Netherlands Publication year 2000 Abstract 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 Subject Biologybeta actincomplementary DNAcrystallinheat shock protein 27heat shock protein 60messenger RNAarticleastrocytomacontrolled studygene expressiongene inductionheat shockpriority journalreverse transcription polymerase chain reactionstandardtechniqueAstrocytomaBinding, CompetitiveBrain NeoplasmsChaperonin 60CrystallinsDNA, ComplementaryHeatHeat-Shock ProteinsHumansNeoplasm ProteinsReagent Kits, DiagnosticReverse Transcriptase Polymerase Chain ReactionRNA, MessengerRNA, NeoplasmStressTumor Cells, Cultured To reference this document use: http://resolver.tudelft.nl/uuid:9e22d31d-d7ec-4623-bde8-3ba038cab1d2 TNO identifier 235367 ISSN 1355-8145 Source Cell Stress and Chaperones, 5 (1), 30-35 Document type article Files To receive the publication files, please send an e-mail request to TNO Library.