Profiling miRNA Expression in Hemiptera

Total RNA (isolated as described before) from adult and nymphal (2^nd—3^rd instars) H. vitripennis was used to study the expression pattern of the conserved and novel microRNAs. The miRNA expression was measured using a two-step process. In the first step, a stem-loop (RT) primer (designed based on previous reports [39 (link)]) was hybridized to the miRNA and reverse transcribed in a pulsed RT reaction [40 (link)]. In the second step, the RT reaction product was PCR amplified using a miRNA specific forward primer and a universal reverse primer (S1 Table) in real time with SYBR green chemistry using a Bio-Rad CFX Real-Time PCR detection system [40 (link)]. Quantification of the relative changes in miRNA expression was performed using the method previously described [40 (link)]. In this relative quantification method, the sample reference was chosen as miR10a and the endogenous control was chosen as ubiquitin [41 (link)]. The data for relative quantities were converted to fold differences by logarithmic transformation to express the data as a normal distribution. The data presented are the averages of three measurements.