Approaches for the Identification and Characterization of RNA–Protein Interactions - Biophysical Approaches to Translational Control of Gene Expression - page 203

10.3.3.4 Preparation of RNA Samples and Microarray Hybridization/

Sequencing

1. Use 50-100 ng RNA to generate labeled cDNA fragments for hybridization to
Affimetrix gene Chip arrays (e.g., Human Tiling 1.0R Array Set (P/N 900774) or
GeneChip Mouse Tiling 1.1R Array Set (P/N 900853)) following the manufac-
turer's protocol. Alternatively, samples may be analyzed by next-generation deep
sequencing.

10.3.4 Notes

1. Harvesting cells when they are ³ 80% con fl uency (1-3 × 10 6 cells) is important to
get sufficiently concentrated total protein (20-50 mg) in the cell lysate.
2. Do not overdry the RNA as it may be difficult to get the nucleic acid back into
solution. If you have trouble resuspending the RNA pellet, incubate at 65 °C for
5 min.
3. We prefer to use random hexamers to prepare cDNA rather than oligo(dT) prim-
ing for several reasons. One key reason is that oligo (dT) will only prime mRNAs,
so rRNA cannot be used as a reference gene. In addition to this, oligo (dT) prim-
ing introduces a 3¢ end bias to samples which could influence the readout.
4. Deep sequencing can be used as an alternative to microarrays for RNA analysis
and is likely to become the method of choice in the near future.

10.4 In Vitro Analyses of RNA-Protein Interactions

In this section we describe three approaches to complement the immunoprecipita-
tion-based approaches outlined above in the analysis of RNA-protein interactions.
These approaches are often useful in validating and extending results obtained from
RNA-protein analyses in tissue culture cells. Importantly, they can also be used as a
discovery tool to identify novel, functionally important RNA-protein interactions.

10.4.1 Electrophoretic Mobility Shift Assays

When purified or recombinant RNA binding proteins are available, RNA
Electrophoretic Mobility Shift Assays (EMSA) or “gel shift” assays are particularly
useful to both confirm the results of co-immunoprecipitation assays as well as to
study biochemical aspects of the RNA-protein interaction—including measuring
binding affinity (by calculating the dissociation constant K d ) and mapping binding
sites (by surveying a series of variant RNA substrates containing deletions/
insertions).