Riboprobes are RNA probes that can be produced by in vitro transcription of cloned DNA inserted in a suitable plasmid downstream of a viral promoter.
Viruses code for their own RNA polymerases, which are highly specific for the viral promoters. Using these enzymes, labeled NTPs, and inserts in both forward and reverse orientations, both sense and antisense riboprobes can be generated from a cloned gene.
Transcription of RNA is performed with the appropriate RNA polymerase (T3, T7 or SP6), depending on the RNA polymerase promoter sites present in the chosen vector. Because these polymerases are extremely promoter-specific (i.e., there is almost no transcriptional cross talk), virtually homogeneous RNA can be obtained using plasmid DNA as the template in a transcription reaction. When it is desirable to copy only insert DNA sequences, the plasmid is linearized at an appropriate restriction site before the transcription reaction and only discrete “run-off” transcripts are obtained, virtually free of vector sequences. RNA transcripts may be used to generate radioactive probes for hybridization to Northern and Southern blots, plaque and colony lifts as well as non-radioactive probes (i.e, labeled with digoxgenin)for in situ hybridization.
Recent references using riboprobes include:
In situ hybridization
- Biswas, S. et al. (2014) Protocadherin-18b interacts with Nap1 to control motor axon growth and aborization in zebrafish. Mol. Biol. Cell. 25,633–42
- Lam, S-Y. et al. (2014) Upregulation of a local renin-angiotensin system in the rat carotid body during chronic intermittent hypoxia. Exper. Physiol.. 99, 220–31.
- Rabot, A. et al. (2014) Interplay of sugar,light and Gibberellins in expression of Rosa hybrida Vacuolar Invertase 1 regulation. Plant Cell Physiol. epub ahead of print.
- Nonaka, A. et al. (2014) Synaptic plasticity associated with memory engram in the basolateral amygdala. J. Neuro. 34, 9305–09.
- Lopez-Garrido, J. et al. (2014) A eukaryotic-like 3´ untranslated region in Salmonella enterica hilD mRNA. Nuc. Acids. Res. 42, 5894–906.
- Zhabokritsky, A. et al. (2014) Pokeweed antiviral protein alters splicing of HIV -1 RNAs resulting in reduced virus production. RNA, 20, 1238–47.
- Vaughan, R. et al. (2014) The Tripartite virions of the brome mosaic virus have distinct physical properties that affect timing of the infection process. J. Vir. 88, 6483–91.
- Yano, K et al. (2013) Multiple rRNA operons are essential for efficient cell growth and sporulation as well as outgrowth in Bacillus subtilis. Microbiology. 159, 2225–36.
- Ko, Chunkyu, et al. (2014) Residues Arg703, Asp777, Arg781of the RNase H domain of Hepatitis B virus polymerase are critical for viral DNA synthesis. J. Vir.. 88, 154–63.
- Campagna, M. et al. (2013) Sulfamoylbenzamide derivatives inhibit the assembly of Hepatitis B Virus Nucleocapsids. J. Vir. 87, 6931–42.
Latest posts by Gary Kobs (see all)
- Characterizing DNA Repair Proteins with Cell-Free Protein Expression - February 5, 2021
- SARS-CoV-2 Nucleocapsid Protein and PA28γ: A Role in Pathogenesis? - December 9, 2020
- More muscle from eggs? Proteo-lipid complex may help prevent age-associated loss of muscle-mass - September 9, 2020