FISH Tag™ RNA Green Kit with Alexa Fluor® 488 dye from Invitrogen

Fluorescence in situ hybridization (FISH) technology permits detection of specific nucleic acid targets within a biological specimen, or in situ meaning where it lies. Targets such as mRNAs expressed in a tissue or genes present on a chromosome can be localized using this technology. Detection of a nucleic acid target in situ is achieved through hybridization of a complementary sequence, fluorescent dye–labeled nucleic acid “probe” to the specimen. Different fluorophores can be used to label different nucleic acid probes for detection of multiple targets simultaneously.

The FISH Tag™ RNA Green Kit with Alexa Fluor® 488 dye from Invitrogen provides a complete workflow solution for fluorescence labeling applications for 10 reactions. It provides all the necessary reagents needed for probe synthesis, labeling, and purification, as well as for imaging the labeled specimen, and includes a very detailed protocol.

I use this kit on a regular basis for the synthesis and fluorescent labeling of RNA probes for downstream applications such as in situ hybridization, or for simple RNA transfection reactions where a fluorescent label is preferred.

One of the advantages of this kit is the complete workflow, starting with the in vitro transcription to incorporate the modified nucleotide, followed by the chemical labeling and the efficient nucleic acid purification technology used for fast purification. After the labeling and the purification of the probe I routinely take a reading on the NanoDrop (Thermo Scientific) at 260 nm as well as at 492 nm. I have found it helpful to use the Invitrogen Dye:Base Ratio Calculator. to calculate the nucleic acid concentration as well as the labeling efficiency (dyes/bases) to enable me to use the labeled probe in an efficient way in downstream applications.

The template picked for in vitro transcription (IVT) should be a construct having either a T7, T3, or SP6 RNA polymerase promoter sequence. Since all of these polymerases are included, it provides the flexibility to pick the promoter which works the best for a given template. Getting started with the IVT is easy. Prepare a master mix and add it to the sample. It is important to remember to add different concentrations of DTT to the master mix depending on what kind of polymerase is used. After the IVT, the sample gets incubated with DNase I and subsequently the amine-modified RNA is purified using a spin column. With the additional precipitation step, trace amounts of free amines, which would interfere with the labeling reaction, are removed. For the labeling, the RNA gets denatured and the labeling reaction is performed in approximately 1 hr. There are fluorophores in different wavelengths available; therefore a multiplex experiment is easy to perform. After an additional column based purification with subsequent precipitation step, the dye-labeled RNA is ready to use. I always perform a 260 nm read on a NanoDrop (Thermo Scientific) with the additional 492 nm read for the dye:base ratio calculation. In the back of the protocol are also suggestions regarding the hybridization procedures being used as well as a detailed troubleshooting paragraph.

Overall, I think the kit works very well, is quick and easy to use.