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Quantitative reverse transcription PCR (qRT-PCR) has been an integral part of characterizing the immunity of  Anopheles  mosquitoes towards  Plasmodium  invasion. Two anti- Plasmodium  factors of  Anopheles , thioester-containing protein 1 (TEP1) and nitric oxide synthase (NOS), play a role in the refractoriness of  Anopheles  towards  Plasmodium  infection and are generally expressed during infection. However, these are less studied in  Anopheles dirus , a dominant malaria vector in Southeast Asia. Furthermore, most studies used a single reference gene for normalization during gene expression analysis without proper validation. This may lead to erroneous quantification of expression levels. Therefore, the present study characterized and investigated the expression profiles of  TEP1  and  NOS  of  Anopheles dirus  during  P. berghei  infection .  Prior to that, the  elongation factor 1-alpha  ( EF1 ),  actin 1  ( Act ) and  ribosomal protein S7  ( S7 ) genes were validated for their suitability as a set of reference genes.  TEP1  and  NOS  expressions in  An. dirus  were found to be significantly induced after  P. berghei  infection.

peerj

Quantitative real-time PCR analysis of Anopheles dirus TEP1 and NOS during Plasmodium berghei infection, using three reference genes