Quantification of Sm-LAMP and Sm-NPC2 Transcripts

Relative abundance of the Sm-LAMP and Sm-NPC2 transcripts was determined for different S. mansoni life cycle stages (eggs, miracidia, cercariae, 5 day old schistosomula, adult males and adult females) and for microdissected digestive and reproductive tissues27 (link) using quantitative real-time PCR (qPCR), to confirm that the selected genes are enriched in the gastrodermis and in life cycle stages within the mammalian host27 (link). Forward and reverse primers (Sigma-Aldrich) were designed for Sm-LAMP and Sm-NPC2 using Primer3 (v.0.4.0) software (Supplementary Table 2). Total RNA samples were DNase treated (Promega) prior to synthesis of cDNA using a QuantiTect® Whole Transcriptome Kit (QIAGEN). All cDNA samples were diluted to 5 ng/μl. Real-time PCR was performed and analysed using previously described protocols61 (link) using DNA segregation ATPase62 (link) (Accession No. Smp176580) as the normalising housekeeping gene. Data were analysed using the Rotor Gene 6 software63 (link).

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Nawaratna S.S., Gobert G.N., Willis C., Mulvenna J., Hofmann A., McManus D.P, & Jones M.K. (2015). Lysosome-associated membrane glycoprotein (LAMP) – preliminary study on a hidden antigen target for vaccination against schistosomiasis. Scientific Reports, 5, 15069.

Publication 2015

Forward and reverse primers DNase treated QuantiTect® Whole Transcriptome Kit

Adult Adult females Cdna Cercariae Digestive Dnase Eggs Gene Housekeeping gene Males Mammalian Npc2 Primers Promega Real time pcr Reproductive Synthesis Transcriptome

Corresponding Organization :

Other organizations : University of Queensland, QIMR Berghofer Medical Research Institute, University of Melbourne, Griffith University

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Variable analysis

independent variables

Life cycle stages of S. mansoni (eggs, miracidia, cercariae, 5 day old schistosomula, adult males, adult females)
Microdissected digestive and reproductive tissues

dependent variables

Relative abundance of the Sm-LAMP and Sm-NPC2 transcripts

control variables

DNA segregation ATPase (Accession No. Smp176580) as the normalising housekeeping gene

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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