Just a general note towards fluorescence anisotropy: Sinve it is quantified via the emission ratio of both measured intensities (parallel and perpendicular to the exciting plane), fluorescence anisotropy does not depend on the overall fluorescence intensity.

Therefore, as along as your total fluorescence intensity is high enough to ensure an adequate measurement error, your anisotropy results won't be negatively influenced by photobleaching.

However, since anisotropy is an additive entity, fluorescein-derivatives derived from your labelled shRNA (e.g. breaking of the covalent linker) will definitely interfere with your measurements by narrowing the assay window. Depending on the extent of "free" fluorophore, it may even cause an assay window of zero...

FITC conjugates are not very stable to elevated temperatures and are very pH sensitive (in terms of their fluoresence behavior; fluoresence yield was found to be higher at pH 7.4 than at pH 6.0 by John N. Lorenz and Eric Gruenstein, A simple, nonradioactive method for evaluating single-nephron filtration rate using FITC-inulin, Am J Physiol Renal Physiol,1999 276:(1) F172-F177). See also the following paper, where FITC-protein conjugates shown poor stability at 37 C compared to other dyes.

Hex, Fam (closely related to FITC), SYBER GREEN, and other dyes used for qPCR and PCR-based sequencing are at least known to survive the temperature cycling you describe. It is good to remember that all dyes are light sensitive and should be kept in the dark as much as possible. Also, if your dye is in the "wrong place" on the RNA, it will or could interfere with protein binding... perhaps that isn't an issue, but you may have to try several dye locations. Are you making any of the RNA by chemical synthesis? If so, you can use C5-modified uridine to introduce dyes in specific locations. These are available from commercial suppliers of DNA oligos. I haven't bought any RNA- we used to make ours chemically, a long time ago. Others can comment on that aspect. Good luck.

Somehow this ref was deleted from my answer above. It describes poor stability of protein-FITC conjugates: Comparison of Three Common Amine Reactive Fluorescent Probes Used for Conjugation to Biomolecules by Capillary Zone Electrophoresis, Peter R. Banks and Donald M. Paquette, Bioconjugate Chemistry 1995 6 (4), 447-458.

I agree with James, FITC is great for many applications. It is a cheap easy to use reagent.

But it is not very stable and tends to bleach.

If it is possible, take your tagged shRNA alone and put it in your reaction conditions.

You could then check your sample for fluorescent in comparison with your stock shRNA.

You can then be sure whether this is your problem.

James and Ehud,

Thank you very much, your answers were very helpful. We will definitely look into more stable conjugates and also whether or not our reaction conditions are destabilizing the FITC tag.

One last question,

Does anyone know the significance of using Mgcl 2+ in fluorescence anisotropy buffers when dealing with nucleic acids? Does it stabilize the nucleic acid structure (in particular RNA)?

Just a general note towards fluorescence anisotropy: Sinve it is quantified via the emission ratio of both measured intensities (parallel and perpendicular to the exciting plane), fluorescence anisotropy does not depend on the overall fluorescence intensity.

Therefore, as along as your total fluorescence intensity is high enough to ensure an adequate measurement error, your anisotropy results won't be negatively influenced by photobleaching.

However, since anisotropy is an additive entity, fluorescein-derivatives derived from your labelled shRNA (e.g. breaking of the covalent linker) will definitely interfere with your measurements by narrowing the assay window. Depending on the extent of "free" fluorophore, it may even cause an assay window of zero...