I am trying to analyze miRNAs using TLDA cards. The basic set-up of my experiment is as follows: I want to identify differentially expressed miRNAs in normal vs tumor samples. From each patient we have collected miRNA from tumor and adjacent normal tissue. For each of these I run 2 TLDA cards, each with 384-wells for a total detection of 768 miRNAs. Each card is run completely separately, sometimes on different days. To analyze the data I want to normalize using the HT-qPCR package. My fundamental confusion stems from how to arrange the data for analysis. For each sample (i.e., tumor from patient A), can I pool together the data from the two cards into a single sample or should each card remain separate? How would pooling the two cards change the results of normalization, if at all?
Thanks in advance,