RNA fluorescence in situ hybridization (FISH), long an indispensable tool for the detection and localization of RNA, is becoming an increasingly important complement to other gene expression analysis methods. Especially important for long noncoding RNAs (lncRNAs), RNA FISH adds the ability to distinguish between primary and mature lncRNA transcripts and thus to segregate the site of synthesis from the site of action.
Researchers from Biosearch Technologies detail a streamlined RNA FISH protocol for the simultaneous imaging of multiple primary and mature mRNA and lncRNA gene products and RNA variants in fixed mammalian cells. The technique makes use of fluorescently pre-labeled, short DNA oligonucleotides (circa 20 nucleotides in length), pooled into sets of up to 48 individual probes. The overall binding of multiple oligonucleotides to the same RNA target results in fluorescent signals that reveal clusters of RNAs or single RNA molecules as punctate spots without the need for enzymatic signal amplification. Visualization of these punctate signals, through the use of wide-field fluorescence microscopy, enables the counting of single transcripts down to one copy per cell. Additionally, by using probe sets with spectrally distinct fluorophores, multiplex analysis of gene-specific RNAs, or RNA variants, can be achieved. The presented examples illustrate how this method can add temporospatial information between the transcription event and both the location and the endurance of the mature lncRNA.
The researchers also briefly discuss post-processing of images and spot counting to demonstrate the capabilities of this method for the statistical analysis of RNA molecules per cell. This information can be utilized to determine both overall gene expression levels and cell-to-cell gene expression variation.
A schematic of the design of inclusive probe sets to detect primary and mature and MYC mRNAs, as well as primary and mature H19, PVT1, and XIST lncRNAs
Noncoding sequences are represented in black and coding sequences in blue. Exons common to all variants are shown as thick lines, alternatively spliced exons as medium thick lines, and introns as thin lines. All four exon probe sets are inclusive and target the common regions of the mature RNA variants. The probe sets are color coded according to the dye label: MYC, H19, and XIST exons with Quasar 570: orange; PVT1 exons with CAL Fluor Red 610: purple; MYC introns with FAM: green; and H19, PVT1, and XIST introns with Quasar 670: dark blue