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Figure 1 | BMC Genomics

Figure 1

From: Stage-specific differential gene expression in Leishmania infantum: from the foregut of Phlebotomus perniciosus to the human phagocyte

Figure 1

Sampling and mRNA amplification. (A) The life cycle of Leishmania spp. (B) Promastigote RNA extraction was performed immediately after dissection of the sand fly guts and mild lysis of U937 cells. Pro-Pper samples were immediately washed in PBS and lysed with TRIzol® for total RNA extraction. After that, mRNA was doubly amplified (aaRNA) due to sample amount requirements. This included two cycles of reverse transcription (RT) plus second strand cDNA synthesis (combining the use of the Klenow fragment and the RNase H) plus in vitro transcription (IVT). The RT reaction of the first amplification round was performed with a poly-dT primer and the second strand synthesis and the RT reaction of the second amplification round were performed with random hexamer primers, all of which were provided in the MessageAmpTM II aRNA Amplification Kit. Three biological replicates were obtained to perform the subsequent microarray experiment. (C) Electrophoresed aaRNA samples used for the microarray analysis after synthesis of labeled cDNA.

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