The amplified fragment was excised from the gel and cDNA was recovered from agarose gel using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol and the purified DNA product was ligated with a pCR 2.1 vector (Invitrogen, Carlsland, CA) according to the manufacturer's protocol to generate the pCR-M5 plasmid. For further confirmation, PCR positive plasmids were sequenced in the DNA Biotechnology Facility of the University of Connecticut. Six micrograms of plasmid DNA was linearized using 6 units of the restriction enzyme Bam HI for 4 h at 37 • C. Then, linearized DNA was used as a template in an in vitro transcription reaction with the T7 RiboMAX Express Large-Scale RNA Production System (Promega, Madison, WI) according to the manufacturer's recommendation. After the in vitro transcription reaction at 37 • C for 1 h, the possible remaining plasmid DNA was digested by DNase I and purified RNA was quantified with a spectrophotometer NanoDrop ND-1000 (Thermo Fisher Scientific, Wilmington, DE). The copy numbers of purified RNA were determined using a previously described method [27] and was used for the generation of a real time standard curve.