Detection of Target mRNAs by Non-RI Hybridization Technique : With Special Reference to Factors Affecting Hybridization Signals

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Factors affecting the Northern blot hybridization signals were examined using synthetic oligonucleotides as probes and the digoxigenin labeling and detection system. The factors were incubation time for labeling probes, substrates for alkaline phosphatase, irradiation intensity of ultraviolet (UV) rays for cross-linking RNA to Nylon membrane, and concentrations of target mRNAs. (1) Labeling efficiency of probes was increased by extending the incubation time with terminal deoxynucleotidyl transferase (TdTase); 15,30,and 60 minutes. The 60-minute incubation produced the labeled probes with longer tails and high detection efficiency. (2) Probes labeled by 60-minute incubation were detected at as low as 10^<-16> mol/μL/dot on Nylon membrane in combination with NBT/BCIP (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate), Vector Blue, or Vector Red for enzyme substrates. Digital images of the signals processed by NIH Image showed the graded sensitivity of the enzyme-mediated precipitations depending on the substrates (NBT/BCIP≧Vector Blue>Vector Red). (3) The intense signals of the Northern blot hybridization were obtained from membranes irradiated by UV rays at 30×10^3 to 120×10^3μJ/cm^2. UV rays at either low or high energy beyond the optimal range reduced the signals. (4) In the Northern blot analysis, poly (A)^+ RNA samples showed high sensitivity for detecting target mRNAs in comparison with total RNA analysis. (5) The protocol established in the present study detected tissue-specific expression of β・γ-actin mRNA, glycerol-3-phosphate dehydrogenase-1 (G3PDH-1) mRNA, preproenkephalin (PPE) mRNA, preprodynorphin (PPD) mRNA in four tissues ; brain, liver, kidney, and testis.
香川県立保健医療大学の論文

Detection of Target mRNAs by Non-RI Hybridization Technique : With Special Reference to Factors Affecting Hybridization Signals

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