Click-iT® RNA Alexa Fluor® 488 Imaging Kit from Invitrogen

The ability to detect newly synthesized RNA or global changes in RNA levels in a temporal manner is an important aspect of molecular biology; important changes in RNA can occur due to disease, environmental damage, or drug treatment.

The Click-iT® RNA Alexa Fluor® 488 Imaging Kit from Invitrogen contains all the components needed to label and detect newly synthesized RNA in whole cells. In addition to the blue fluorescent Hoechst 33342 dye as a nuclear counterstain for DNA profiling, the kit includes sufficient reagent for labeling 25, 18 x 18 mm coverslips using a 500 ul reaction volume per well. For my experiments, I used fixed cells, which were spun down on coverslips using a cyto-spin (Shandon, Thermo Scientific); therefore, the reaction volume I needed was much smaller and the kit lasted significantly longer. The kit is also available with the Alexa Fluor® 594 dye if this is more convenient due to other co-staining procedures used.

In my hands, the kit got used for monitoring de novo RNA expression in mammalian cells using laser confocal microscopy under different stimulatory conditions. The reactions were performed with paraformaldehyde (PFA) fixed and permeabilized cells, following the protocol recommendations. The cells were spun down on glass coverslips.

Since the kit utilizes an alkyne-modified nucleoside (EU), which gets incorporated into RNA but not DNA, newly synthesized RNA can be detected without the use of radioactivity or antibodies. There are several advantages of the technique used in this kit over traditional detection methods. The detection reaction is based on a chemoselective ligation which happens quickly and efficiently. In addition, the copper catalyzed reaction is bioinert and no side reactions occur; therefore, it is highly sensitive and results in a low background staining. Due to its staining characteristics, it is possible to use additional labels for multiplex analysis.

The experimental approach I used was based on the general protocol which comes with the kit, and is easy and straightforward. The cells were incubated with the alkyne–nucleoside EU, after preparation of an EU stock solution with deonized water. The cells were fixed using 2% PFA. The company recommends 3.7%, but with my standard PFA fix everything worked quite well. The next step was a permeabilization using standard reagents. During permeabilization, there was enough time to prepare the reaction mix. In my opinion, this is the most critical step, since the reaction buffer needs to be prepared fresh and used immediately after preparation due to unwanted oxidation reactions. After the 30 minute incubation period, the samples were washed using the included rinse buffer. All these steps need to be done while the specimens are protected from light. After the rinsing step, cells are ready for visualization or co-staining procedures. In this regard, the protocol includes a list of substances which are compatible with the kits chemistry. For my expeiments I used wheat germ agglutinin 555 (Invitrogen) which stains sialic acids as a costain. The stained cells were visualized using laser confocal microscopy.

One thing I was missing while using he kit was the inclusion of a transcription inhibitor as a negative control, which would have been a nice additional feature.

I think the kit is a great tool, especially if you want to show RNA synthesis by means of microscopy.