Assessment of the Specificity of A Hybridization of Surfactant Protein A by Addition of Non-specific Rat Spleen RNA

Background Nucleic acid hybridization has become an essential technique in the development of our understanding of gene structure and function. The quantitative analysis of hybridization has been used in the measurement of genome complexity and gene copy number. The filter hybridization assay is rapid, sensitive and can be used to measure RNAs complementary to any cloned DNA sequence. Methods The authors assessed the accuracy, linearity, correlation coefficient and specificity of the hybridization depending on the added dose(0, 1, 5, and 10 µg) of non-specific rat spleen RNA to hybridization of surfactant protein A mRNA. Filter hybridization assays were used to obtain the equation of standard curve and thereby to quantitate the mRNA quantitation. Results 1. Standard curve equation of filter hybridization assay between counts per minute (X) and spleen RNA input (Y) was Y=0.13X-19.35. Correlation coefficient was 0.98. 2. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) was Y=0.00066X-0.046. Correlation coefficient was 0.99. 3. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) after the addition of 1 µg spleen RNA was Y=0.00056X-0.051. Correlation coefficient was 0.99. 4. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) after the addition of 5 µg spleen RNA was Y=0.00065X-0.088. Correlation coefficient was 0.99. 5. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) after the addition of 10 µg spleen RNA was Y=0.00051X-0.10. Correlation coefficient was 0.99. Conclusions Comparison of cpm/filter in a linear range allowed accurate and reproducible estimation of surfactant protein A mRNA copy number irrespective of the addition dosage of non-specific rat spleen RNA over the range 0-10 µg.