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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

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Author: Bajramović, J.J. · Geutskens, S.B. · Bsibsi, M. · Boot, M. · Hassankhan, R. · Verhulst, K.C. · Noort, J.M. van
Type:article
Date:2000
Institution: Div. Immunological and Infect. Dis., TNO Prevention and Health, PO Box 2215, 2301 CE Leiden, Netherlands
Source:Cell Stress and Chaperones, 1, 5, 30-35
Identifier: 235367
Keywords: Biology · beta actin · complementary DNA · crystallin · heat shock protein 27 · heat shock protein 60 · messenger RNA · article · astrocytoma · controlled study · gene expression · gene induction · heat shock · priority journal · reverse transcription polymerase chain reaction · standard · technique · Astrocytoma · Binding, Competitive · Brain Neoplasms · Chaperonin 60 · Crystallins · DNA, Complementary · Heat · Heat-Shock Proteins · Humans · Neoplasm Proteins · Reagent Kits, Diagnostic · Reverse Transcriptase Polymerase Chain Reaction · RNA, Messenger · RNA, Neoplasm · Stress · Tumor Cells, Cultured

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