The group of 4 that amplified first was the females (prepared on 12-15 with []'s of 512, 367, 553, and 455ng/ul from the bioanalyzer) while the second group of 4, amplifying around 14 cycles is the males (prepared on 12-7 with []'s of 3086, 5631, 1783, 2363ng/ul from the bioanalyzer). The initial concentrations shouldn't matter much because I dilute them down to have 2ng of RNA entering the ssTruseq protocol. In this case though, it seems that I over-diluted the males causing them to amplify later. There is something really odd going on here because when I looked back through my lab book, the concentration of those samples from the nanodrop was much less: 1351, 1559, 1027, and 1276ng/ul. In fact these concentrations are ~4x less (i.e.: two pcr cycles different...). As they amplify on average 2 cycles later, I took a look back at the bioanalyzer results and found that the concentrations were slightly outside the optimal range and therefore likely a little spurious.
In the future I will dilute my samples down and then nano-drop them before I start the ssTruSeq protocol in order to avoid starting with different concentrations of RNA.
----------------------------------------------------------------------------------------------------------------------------
Sultan, M., S. Dökel, V. Amstislavskiy, D. Wuttig, H. Sültmann, H. Lehrach and M.-L. Yaspo. 2012. A simple strand-specific RNA-Seq library preparation protocol combining the Illumina TruSeq RNA and the dUTP methods. Biochemical and Biophysical Research Communications 422:643–646. Elsevier Inc.
No comments:
Post a Comment