Hi, I recently performed RNA-seq experiment with ERCC spike-ins. I added the recommended amounts of mix1 to WT and mix2 to mutant total RNA before library preparation. I calculated the observed ratios (log2 RPKM >=1) vs expected ratios and found that the R2 value about 0.90 but the slope is ~0.7. This indicates that the observed ratios are some what off to expected ratios. I do not understand whether I can use the slope from equation to normalize the reads for all the genes?
I also plotted the log2 RPKM values against their expected concentrations and also found good correlation (0.95) but the slope is 0.77.
Can any explain whats happening and whether I can use this for normalization of the genes?