Nonisotopic detection of microRNA using digoxigenin labeled RNA probes.
MicroRNAs (miRNAs) are an important class of endogenously derived, small approximately 22 nucleotide noncoding regulatory RNAs that have recently become implicated in development, cell regulation and cancers of various tissues. Here we report a nonisotopic Northern analysis method for miRNA detection using 3'-digoxigenin (DIG)-labeled RNA oligo probes. Northern blot analysis was performed using miRNA or total RNA fractions extracted from human leukemic cell lines, and blots were hybridized with either 32P- or DIG-labeled RNA probe for miR-181, miR-155 or miR-16. A labeled probe for U6 small nuclear RNA served as an internal control. The use of DIG-labeled RNA probes was equally sensitive compared to 32P-labeled probes in detecting miRNA quantities as low as 50 ng. The ability to use nonisotopic methods and yet obtain sensitive and reliable results offers an advantage to investigators who prefer to avoid isotopes.
Ramkissoon
Shakti H
SH
Howard Hughes Medical Institute-National Institutes of Health Research Scholars Program, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Bethesda, MD, USA.
Mainwaring
Lori A
LA
Sloand
Elaine M
EM
Young
Neal S
NS
Kajigaya
Sachiko
S
D015152:Blotting, Northern / Q000379:methods*; D002460:Cell Line; D004076:Digoxigenin / Q000378:metabolism*; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism*; D015394:Molecular Structure; D009693:Nucleic Acid Hybridization; D015347:RNA Probes / Q000737:chemistry / Q000378:metabolism*
D035683:MicroRNAs; D015347:RNA Probes; D004076:Digoxigenin