Nonradioactive Northern blotting with biotinylated and digoxigenin-labeled RNA probes.

The application of nonradioactive RNA probes for Northern blotting offers the advantage of a rapid turn-around time for results without the loss of sensitivity for target mRNA detection. However, a problem that has impeded the widespread use of nonradioactive RNA probes for use in Northern blotting is the difficulty in stripping these probes from nylon membranes after hybridization. In this report we describe two protocols for stripping digoxigenin (Dig)-labeled RNA probes from nylon membranes. One protocol utilizes a phosphate-buffered formamide stripping solution to remove nonchemically modified (regular) RNA probes while the other method utilizes strippable probes that were produced with a chemically modified nucleotide (CTP) and removed by a specific stripping solution. This latter method was developed by Ambion Inc. and is called Strip-EZ. We also describe a protocol for the detection of two separate rat mRNAs using both biotin and digoxigenin-labeled RNA probes that does not require stripping the membrane after hybridization. Finally, we describe the use of another new labeling technology, called Chem-Link, that quickly and conveniently labels RNA for use in Northern blotting.

Biotin in vitro translation, nonradioactive detection of cell-free synthesized proteins.

In vitro translation of mRNAs into proteins is frequently used to study the coding capacity of RNAs or cDNAs and the functional effects of mutations. In vitro translation assays have traditionally been monitored by following the incorporation of a radiolabeled amino acid into newly synthesized protein. We have optimized an alternative nonradioactive biotin-labeling method. tRNALys is first aminoacylated with lysine, which is then chemically labeled with biotin. When biotin-lysine-tRNALys is added to translation systems, the biotinylated lysine is incorporated into the growing polypeptide chain. After electrophoresis and transfer to a blotting membrane, the biotin-labeled translation products are detected by a chemiluminescent reaction of luminol/iodophenol with streptavidin-coupled horseradish peroxidase. This nonradioactive method yields results equivalent to those obtained using the radioactive method. Biotin-labeled translation products are also biologically functional: (i) biotinylated precursor proteins are transported and processed correctly by dog pancreas microsomes; (ii) transcription factors synthesized by biotin in vitro translation bind specifically to their DNA recognition sequence; and (iii) biotin-modified luciferase keeps its enzymatic activity. The major advantage of the biotin in vitro translation system is that no radioactivity is required, and the method is easy, economical, reproducible and fast--the whole nonradioactive procedure, from translation to detection, can be completed within six hours.

Multiple nucleic acid labeling and rainbow detection.

A method which allows discrete nucleic acid sequences to be detected with differently colored hybridization signals on the same blot involving only a single hybridization step is described. Nucleic acid probes labeled with digoxigenin, fluorescein, or biotin are hybridized simultaneously to immobilized target nucleic acids. Differential colorimetric detection is carried out in consecutive alkaline phosphatase-based immunoassays with one of three 3-hydroxy-2-naphthoic acid anilide phosphate/diazonium salt combinations as substrate. Each label is visualized by a different color precipitate (green, red, and blue) directly on the membrane. We demonstrate the use of this method in multicolor plasmid mapping, detection of different genomic sequences on a single Southern blot, discrimination of transcription levels in a Northern blot, and colony screening. Advantages and limitations of the method, as well as further applications, are discussed.