I am trying to determine the cellular localisation of a lncRNA in THP1 cells. To do this, I have fractionated cells so that I have Cytoplasmic, Nucleoplasmic and Chromatin Associated fractions.
My questions is, how do I normalise the data, as housekeeping genes are not expressed at equal levels across the different fractions? I am concerned that as my housekeeping gene varies across the compartments, this will introduce an artefact in the analysis, making it seem as though my lncRNA of interest is enriched in a certain compartment.
Thank you!
I guess you could use compartment specific housekeeping genes so that your analysis won't be biased.
I am also having the same confusion. Can someone please let us know that which gene can be used as a positive control for nuclear fraction and which gene for the cytosolic fraction. I have got some suggestions for nuclear fraction i.e. using U6 gene as a positive control for nuclear and negative for cytosolic. Can we use tRNA gene as a positive control for cytosolic and negative for nuclear fraction? Please someone give details about this. It would be great help. Thanks in advance.
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