Improvement on the extraction method of RNA in mites and its quality test

To solve the long-existing difficult problems in extracting RNA and constructing a complementary DNA (cDNA) library for trace mites, we conducted a further comparative experiment among three RNA extraction methods (TRIzol method, Omega method, and Azanno method) based on our previous attempts at the construction of cDNA library of mites, with Psoroptes cuniculi still used as the experimental subject. By subsequently decreasing the number of mites, the least number of mites needed for RNA extraction of each method were found by criteria of completeness, concentration, and purity of the extracted RNA. Specific primers were designed according to the allergen Pso c1, Pso c2, and Actin gene sequences of Psoroptes to test the reliability of cDNA library. The results showed that Azanno method needed only 10 mites with sensitivity 204 times higher than previously used TRIzol method and 20 times higher than Omega method; clear RNA band was detected by agarose gel electrophoresis; and ultraviolet spectrophotometer determination showed that RNA concentration, 260/280, and 260/230 were in the range of 102 to 166 ng/μl, 1.83 to 1.99, and 1.49 to 1.72, respectively. Finally, specific primers detection showed that the amplified sequences had 98.33, 98.19, and 99.52% identities with those of P. cuniculi or Psoroptes ovis in GenBank, respectively, indicating that the cDNA library constructed using 10 mites was successful and it could meet the requirements for molecular biology research. Therefore, we concluded that Azanno method was more effective than TRIzol method and Omega method in RNA extraction and cDNA library construction of trace mites.