Storage phosphor imaging technique improves the accuracy of RNA quantitation using 32P-labeled cDNA probes.
The use of the storage phosphor imaging technique to quantitate radioactivity on blots generated by hybridization with 32P-cDNA probes was evaluated and compared with screen-enhanced x-ray film autoradiography. Quantitation of RNA dot blots hybridized with a 28S ribosomal RNA-specific cDNA probe showed that storage phosphor imaging was more sensitive than screen-enhanced x-ray film autoradiography in identifying low amounts of total RNA (1-10 micrograms). Evaluation of Northern blots containing 30 micrograms of total RNA from human skin biopsies hybridized with a cDNA probe for the human acidic ribosomal phosphoprotein, PO, showed that both techniques detected random biological variability of this housekeeping gene in a similar manner. The two techniques exhibited a strong linear correlation in their ability to quantitate mRNA levels of a retinoic acid-inducible gene (RIS-1). This correlation was stronger at levels corresponding to 1-fold to 30-fold increases of signal and decreased beyond this range because of the insensitivity of the x-ray film. In conclusion, storage phosphor imaging is more accurate than screen-enhanced x-ray film autoradiography in identifying different RNA amounts (higher sensitivity) and in detecting increasing RNA signals at high levels of radioactivity (higher dynamic range).
Zouboulis
C C
CC
University of Michigan, Ann Arbor.
Tavakkol
A
A
D000818:Animals; D001345:Autoradiography / Q000379:methods; D001709:Biotechnology; D018076:DNA, Complementary; D006801:Humans; D051379:Mice; D015336:Molecular Probe Techniques / Q000706:statistics & numerical data; D010761:Phosphorus Radioisotopes; D012313:RNA / Q000032:analysis* / Q000235:genetics*; D011856:Radiographic Image Enhancement; D012680:Sensitivity and Specificity; D014962:X-Ray Film
D018076:DNA, Complementary; D010761:Phosphorus Radioisotopes; D012313:RNA