Dot-blot hybridization: quantitative analysis with direct beta counting.
The suitability of using direct beta counting (DBC) for quantitating radioactivity of the probe:target complex in dot-blot hybridization was evaluated using a Packard Matrix 96. A comparison of blots analyzed using autoradiography followed by densitometry scanning (film/densitometry) with those analyzed using direct beta counting revealed similar data trends with the two methods. However, direct beta counting quantitated the amount of radioactivity in the dot blots directly (without film exposure or additional sample preparation), which significantly reduced the time required to obtain results. Blots analyzed first with direct beta counting and then liquid scintillation counting exhibited similar data trends with both methods. Despite a decreased counting efficiency, analysis with direct beta counting has the following advantages compared with liquid scintillation counting: 1) no additional sample preparation is required (no vials or cocktail are used), 2) no sample destruction occurs due to analysis and 3) quantitative results are obtained more rapidly (since the radioactivity for all 96 samples in a dot blot is simultaneously determined in real time). Analysis with direct beta counting was also shown not to interfere with the successful reprobing of stripped dot blots with either unique sequence or total genomic probes. Overall, direct beta counting provides quick, quantitative results for dot blots while saving considerable time and effort.
Sognier
M A
MA
Department of Radiation Therapy, University of Texas Medical Branch, Galveston 77550.
Neft
R E
RE
Roe
A L
AL
Eberle
R L
RL
Belli
J A
JA
D000818:Animals; D001610:Beta Particles; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000032:analysis; D003720:Densitometry; D005069:Evaluation Studies as Topic; D007700:Kinetics; D015336:Molecular Probe Techniques; D009693:Nucleic Acid Hybridization; D010761:Phosphorus Radioisotopes; D011874:Radiometry / Q000379:methods*
D010761:Phosphorus Radioisotopes; D004247:DNA