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

Area of Science:

• Molecular Biology
• Biochemistry
• Analytical Chemistry

Background:

• Accurate gene quantification is crucial for understanding biological processes.
• Competitive reverse transcriptase polymerase chain reaction (RT-PCR) is a common method for gene quantification.
• Traditional analysis methods like agarose gel electrophoresis may miss important reaction products.

Purpose of the Study:

• To evaluate the utility of ion-pair reversed-phase High-Performance Liquid Chromatography (HPLC) for gene quantification using competitive RT-PCR.
• To determine if HPLC can improve the accuracy of gene quantification by detecting all reaction products, including heteroduplexes.

Main Methods:

• Competitive RT-PCR was performed using RNA competitors with high sequence similarity to native transcripts.
• Reaction products were analyzed using both agarose gel electrophoresis and ion-pair reversed-phase HPLC.
• Mathematical analysis was employed to quantify reaction products, including heteroduplexes.

Main Results:

• HPLC detected three products, including a heteroduplex, while gel electrophoresis detected only two.
• Quantification of heteroduplexes was essential for accurate gene quantification.
• PCR amplification efficiency was similar for native and competitor transcripts, but RT efficiency varied by sequence.
• Increasing sequence similarity improved RT efficiency consistency.
• The assay demonstrated high accuracy, reflecting actual gene copy numbers.
• The method was adapted to quantify alternatively spliced isoforms.

Conclusions:

• Ion-pair reversed-phase HPLC significantly improves gene quantification accuracy in competitive RT-PCR by enabling heteroduplex detection.
• This technique offers a precise and accurate method for measuring gene expression and analyzing alternative splicing.
• The developed assay system is reliable for determining absolute gene copy numbers and relative isoform abundance.