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
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
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TNO identifier
235367
ISSN
1355-8145
Source
Cell Stress and Chaperones, 5 (1), 30-35
Document type
article