Dramatically improved RNA in situ hybridization signals using LNA-modified probes.
In situ detection of RNA by hybridization with complementary probes is a powerful technique. Probe design is a critical parameter in successful target detection. We have evaluated the efficiency of fluorescent DNA oligonucleotides modified to contain locked nucleic acid (LNA) residues. This increases the thermal stability of hybrids formed with RNA. The LNA-based probes detect specific RNAs in fixed yeast cells with an efficiency far better than conventional DNA oligonucleotide probes of the same sequence. Using this probe design, we were also able to detect poly(A)(+) RNA accumulation within the nucleus/ nucleolus of wild-type cells. LNA-based probes should be readily applicable to a diverse array of cells and tissue samples.
Thomsen
Rune
R
Centre for mRNP Biogenesis and Metabolism, Department of Molecular Biology, Aarhus University, Denmark.
Nielsen
Peter Stein
PS
Jensen
Torben Heick
TH
D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000235:genetics*; D011061:Poly A / Q000737:chemistry* / Q000378:metabolism; D011062:Polynucleotide Adenylyltransferase / Q000235:genetics / Q000378:metabolism; D012331:RNA, Fungal / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D018840:HSP70 Heat-Shock Proteins; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D012331:RNA, Fungal; C456269:SSA4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C477371:locked nucleic acid; D011061:Poly A; C429559:PAP1 protein, S cerevisiae; D011062:Polynucleotide Adenylyltransferase; D004259:DNA-Directed DNA Polymerase; C487545:PAP2 protein, S cerevisiae