Figure 3

5' Rapid amplification of cDNA ends (RACE) analysis of kallikrein 1b26 (klk1b26) mRNAs from male and female submandibular glands (SMGs) and effect of incubation with SMG extracts on the mRNA. (A) Antisense chains of 5' RACE products were prepared and analyzed as described in Methods. Each signal is indicated by color: green = A; red, = T; black = G; blue = C. (B) Degradation of klk1b26 mRNA by incubation with the SMG extracts. Total RNA from male SMGs (21.5 μg) was incubated at 37°C for 30 min with an extract prepared from male or female mouse SMGs as described in Methods. After the incubation the total RNA was again purified with the Micro-to-Midi Total RNA Purification System (Invitrogen) and was eluted to 20 μl, quantitatively. First-strand DNAs were prepared with 280 ng of the total RNAs thus treated with SMG extracts and reverse transcription (RT)-PCR was carried out by using primer pairs F21/R552 or F169/R552. Representative results are shown. Similar results were obtained in three independent experiments. Quantitative determination of density of the PCR products was made by computer-assisted image analysis (NIH image; http://rsbweb.nih.gov/nih-image/). Relative densities of the PCR signals with total RNAs treated by 8 μg/μl each of male SMG extract and female SMG extract were estimated to be 7.36 ± 0.40 and 7.44 ± 0.51, respectively (mean ± SD; n = 3 each). There was no significant difference in the degrading activities for the mRNA between the male and female SMG extracts.