Quantification of Differential RNA-Binding
XRNAX followed by silica enrichment can not only be used for the discovery of RNA-binding proteins but also to assess RNA-binding differentially between conditions. Therefore cells of one SILAC label undergo treatment, whereas cells of the complementary SILAC label serve as a control. Both populations are UV-crosslinked, combined and subjected to the identical workflow as used for the discovery of RNA-binding proteins. Consequently, SILAC ratios between the two samples quantify relative RNA-binding of hundreds of proteins between the two conditions.
Learn here about the proteome-wide quantification of differential RNA-binding starting from XRNAX extracts (refer to The Protocol section for initial XRNAX extraction). Note that the protocol downstream of the XRNAX extraction is identical to the one for the exploration of RNA-binding proteomes. However, data analysis for the differential quantification requires certain attention. Please refer to the technical note at the bottom of this protocol for details.
For the quantification of differential RNA-binding use 1000 µg of XRNAX extract (2 confluent 245 x 245 mm2 dishes MCF7 cells yield approximately 1000 µg XRNAX extract). Culture cells with two SILAC labels and treat them differentially (e.g. SILAC light: no treatment control, SILAC heavy: treatment). UV-crosslink and harvest both populations as described in the ‘The Protocol’ section. Combine the two populations during the initial TRIZOL lysis step of XRNAX and proceed without further changes.
Downstream Protocol for the Quantification of Differential RNA-Binding
Use the identical protocol as described for the exploration of RNA-binding proteomes at the top of this page.
Technical Note Concerning SILAC Quantification
The above scheme illustrates how silica enrichment is used to remove non-crosslinked protein before SILAC quantification. Like every enrichment method silica enrichment comes with a certain background. Effectively this means that some peptides do not show quantitative behavior in the SILAC-based quantification. Using a non-UV-crosslinked control allows for the identification of peptides, which do not show any background at all and therefore exhibit quantitative behavior in the normal dynamic range of SILAC (approx. two magnitudes of order). Our experiments on the three cell lines MCF7, HEK293 and HeLa have identified over 18000 of those peptides. The list of these peptides is provided here as a download:
Make sure to filter your results for only those peptides so that your assessment does not get skewed by background. Alternatively, introduce a third SILAC label serving as a non-UV-crosslinking control, which will directly flag background peptides. We do no recommend this solution as our experiments indicate that a third SILAC label reduces the number of quantified peptides significantly.