Quantitative analysis of gene expression by ion-pair high-performance liquid chromatography

Journal of Chromatography. a
P A DorisA Hayward-Lester

Abstract

We have analyzed the utility of ion-pair reversed-phase HPLC for gene quantification by competitive reverse transcriptase polymerase chain reaction (RT-PCR). Competitive RT-PCR reactions employed various RNA competitors which shared high sequence similarity to the native transcripts for which they served as references. Competitive reactions resulted in the detection of two reaction products when reactions were analyzed by agarose gel electrophoresis, but three products when analyzed by HPLC. The third product was demonstrated to be a heteroduplex formed between mixed strands of native and competitor amplicons. Mathematical analysis of these competitive reactions indicated that identification and quantification of the heteroduplexes were essential to produce accurate gene quantification. PCR amplification efficiency was shown to be identical for native and competitor transcripts. However, RT efficiency differences were observed which may be sequence dependent. These differences were highly consistent across reactions for the same native and competitor inputs. Increasing the sequence similarity resulted in a competitor which had the same RT efficiency as the native transcript. Titration of various levels of competitor against nat...Continue Reading

References

Jan 1, 1992·Electrophoresis·S NathakarnkitkoolG K Bonn
Oct 8, 1992·Nature·P D Siebert, J W Larrick
Apr 1, 1990·Proceedings of the National Academy of Sciences of the United States of America·G GillilandH F Bunn
Dec 1, 1989·Proceedings of the National Academy of Sciences of the United States of America·A M WangD F Mark
Mar 1, 1983·Endocrinology·X Z ShenS L Woo
Nov 15, 1994·Analytical Biochemistry·P J OefnerG K Bonn
Sep 1, 1994·Journal of Endocrinological Investigation·L MieleA B Mukherjee
Nov 1, 1993·Analytical Biochemistry·L Raeymaekers
Apr 2, 1996·Proceedings of the National Academy of Sciences of the United States of America·G C KunduA B Mukherjee
Aug 1, 1995·Genome Research·L Raeymaekers

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Citations

Nov 30, 2002·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Peter J Oefner, Christian G Huber
Jun 20, 2003·Human Mutation·Laura CremonesiPaolo Arosio
Apr 25, 2000·Hypertension·D B Kainer, P A Doris
Jun 1, 2001·Human Mutation·W Xiao, P J Oefner
Sep 27, 2018·The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences·Ditte SøgaardJørn Wulff Helge
Mar 21, 2012·The FEBS Journal·Halina CarDiana Sawicka
Apr 14, 2011·Journal of Lipid Research·Takuji NabetaniYoshio Hirabayashi

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