Analysis of var Gene Transcript Patterns by Quantitative Real-Time PCR

Abstract

Quantitative real-time PCR (qPCR) is a simple and sensitive method for determining the amount of a specific target DNA sequence present in a sample. Compared to RNA-seq, reverse transcription qPCR (RT-qPCR) is fast, requires only low input material and is easy to analyze. Therefore, qPCR is widely used to analyze gene expression in P. falciparum, including analyses of the multicopy gene families encoding variant surface antigens (VSAs), whose expression is clonally variant and prone to changes over time. In the recent years, several P. falciparum genomes of culture-adapted strains have been sequenced, providing the knowledge to design variable gene family-specific qPCR primers for each P. falciparum genetic background. Here, we describe the required materials, methods and key factors to perform RT-qPCR experiments to determine VSA transcript abundances in the P. falciparum clones 3D7/NF54, IT4, HB3, and 7G8.

Keywords: Gene transcription analysis; Multicopy gene families; PfEMP1; Quantitative real-time PCR; Variant surface antigens; Virulence genes; qPCR.