Trypanosoma cruzi mini-exon based mRNA amplification technique

We have developed a method of mRNA amplification based on T7 polymerase, where T7 promoter is attached to a oligonucleotide containing the mini-exon sequence, which will hybridize with cDNA molecules containing the anti-ME sequence The method was tested on two different stages of T. cruzi (epimastigotes, Epi and cell-derived trypomastigotes, Trp) and for each one we have two biological replicates (A and B). Each mRNA sample was processed in three distinct ways: poly-A purification (polA), oligo-dT T7 (dT) amplification and oligo-ME T7 (ME) amplification. For the other analyzes, one biological replicate of epimastigotes (EpiA) was selected and processed for the following series of experiments: mRNA quantity (100ng, 50ng, 25ng, 12.5ng, 6.25ng and 3.125ng); mixed sample with human mRNA (mRNA from HeLa cells, in the following masses (T. cruzi / Human): 100ng/0ng (100%), 100ng/900ng (10%), 10ng/990ng (1%), 5ng/4995ng (0.1%), 10ng/9990ng (0.1%). For the majority of the mixed sample series, Pfx was used as enzyme for 2nd strand synthesis. EpiB was used for mRNA extraction after cell sorting. All samples were processed in two technical replicates (1 and 2). For the cell sorting, there is three technical replicates. There is no technical replicates for the Poly-A samples