Semiquantitative reverse transcription-polymerase chain reaction with the Agilent 2100 Bioanalyzer
- 1 October 2001
- journal article
- research article
- Published by Wiley in Electrophoresis
- Vol. 22 (18) , 4016-4022
- https://doi.org/10.1002/1522-2683(200110)22:18<4016::aid-elps4016>3.0.co;2-9
Abstract
We have applied a method to monitor mRNA expression in a semiquantitative fashion on the Agilent 2100 Bioanalyzer. The method was originally described in 1994 by Wong et al. and referred to as the „primer‐dropping”︁ method. This polymerase chain reaction (PCR) technique uses multiple sets of primer pairs in a coamplification reaction that amplifies the target of interest within a predetermined range specific for each target. Separation, detection and quantification of PCR products were accomplished using the Agilent 2100 Bioanalyzer in conjunction with the DNA 500 and the DNA 1000 LabChip kits for the detection of DNA fragments with a maximum size of 500 and 1000 bp, respectively. Using primers specific for the inducible form of hsp72 and primers for glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as an internal standard we were able to rapidly monitor and quantify inducible hsp72‐mRNA expression.Keywords
This publication has 13 references indexed in Scilit:
- Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.Genome Research, 1995
- Monitoring mRNA Expression by Polymerase Chain Reaction: The "Primer-Dropping" MethodAnalytical Biochemistry, 1994
- Quantitation of cytokine mRNA levels utilizing the reverse transcriptase-polymerase chain reaction following primary antigen-specific sensitization in vivo—I. Verification of linearity, reproducibility and specificityMolecular Immunology, 1991
- Determining transcript number using the polymerase chain reaction:Pgk-2,mP2, andPGK-2transgene mRNA levels during spermatogenesisNucleic Acids Research, 1991
- Use of the polymerase chain reaction in the quantitation of MDR-1 gene expressionBiochemistry, 1990
- Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction.Proceedings of the National Academy of Sciences, 1990
- Measurement by quantitative PCR of changes in HPRT, PGK-1, PGK-2, APRT, MTase, and Zfy gene transcripts during mouse spermatogenesisNucleic Acids Research, 1990
- Quantitation of mRNA by the polymerase chain reaction.Proceedings of the National Academy of Sciences, 1989
- Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by aPCR aidedtranscipttitration assay (PATTY)Nucleic Acids Research, 1989
- Wound Macrophages Express TGF-α and Other Growth Factors in Vivo: Analysis by mRNA PhenotypingScience, 1988