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
article
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
Topics
beta 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
TNO Identifier
235367
ISSN
13558145
Source
Cell Stress and Chaperones, 5(1), pp. 30-35.
Pages
30-35
Files
To receive the publication files, please send an e-mail request to TNO Repository.