Hybridization of biotinylated oligo(dT) for eukaryotic mRNA quantitation.
Quantitation of mRNA content in samples of total cellular RNA is required for the analysis of Northern blot hybridization to estimate the relative level of specific gene expression. Commonly used methods based on UV absorbance and dye staining measure only total RNA, and mRNA normalization by probing for mRNA levels of housekeeping genes, such as beta-actin and glyceraldehyde-3-phosphate dehydrogenase, assumes a constant level of their expression, which, in fact, may vary as a function of cell proliferation and differentiation. We describe here a nonradioactive, slot-blotting method for quantifying eukaryotic mRNA levels using a biotinylated oligo(dT) probe, which hybridizes directly to the 3'-polyadenylated sequence of eukaryotic mRNAs. The method provides a more accurate estimation of mRNA content in total RNA samples and should be applicable for quantitative Northern analysis.
Stolz
L E
LE
Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Tuan
R S
RS
D001710:Biotin; D015152:Blotting, Northern / Q000379:methods*; D005057:Eukaryotic Cells; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000032:analysis*
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D001710:Biotin