Stolz L E, Tuan R S
Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Mol Biotechnol. 1996 Dec;6(3):225-30. doi: 10.1007/BF02761704.
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.
为了分析Northern印迹杂交以估计特定基因表达的相对水平，需要对总细胞RNA样品中的mRNA含量进行定量。常用的基于紫外吸收和染料染色的方法只能测量总RNA，通过检测管家基因（如β-肌动蛋白和甘油醛-3-磷酸脱氢酶）的mRNA水平来进行mRNA标准化，假定它们的表达水平恒定，而实际上其表达水平可能会随细胞增殖和分化而变化。我们在此描述一种使用生物素化寡聚（dT）探针定量真核mRNA水平的非放射性狭缝印迹法，该探针可直接与真核mRNA的3'-聚腺苷酸化序列杂交。该方法能更准确地估计总RNA样品中的mRNA含量，适用于定量Northern分析。
