Differential Evolution of Antiretroviral Restriction Factors in Pteropid Bats as Revealed by APOBEC3 Gene Complexity

Hayward, Joshua A; Tachedjian, Mary; Cui, Jie; Cheng, Adam Z; Johnson, Adam; Baker, Michelle L; Harris, Reuben S; Wang, Lin-Fa; Tachedjian, Gilda

To confirm A3 gene product expression, polymerase chain reaction (PCR) amplification assays were performed using cDNA generated from P. alecto spleen tissue and various combinations of forward and reverse primers designed using the A3 transcript predictions identified in the transcriptome analysis. P. alecto trapping, tissue collection and RNA extraction was performed as reported previously (Cowled et al. 2011 ) except RNAlater (Ambion) preserved spleen from four male adult bats was pooled prior to tissue homogenization followed by total RNA extraction with the Qiagen RNeasy Mini kit with on-column removal of genomic DNA with DNase I. Total RNA was reverse transcribed into cDNA with the Qiagen Omniscript reverse transcriptase according to the manufacturer's protocol except that the reaction contained 100 ng/ll total RNA, 1 lM oligo(dT) 18 (Qiagen), and 10 lM random hexamers (Promega).