Rapid Amplification of cDNA Ends for Encephalitozoon

5′ cDNA ends were characterized with the SMARTer RACE Amplification kit (Clontech Laboratories, Inc., Mountain View, CA, USA) according to the manufacturer’s recommendations. The reverse transcription (RT) reaction step was performed with 200 ng of E. cuniculi total polyuridylated RNAs using a universal poly(A)-stem-loop RT primer.³³ (link) This first strand reaction products were diluted with 50 µl of tricine-EDTA buffer and used both for 5′ RACE-PCR (0.2 µM of each specific primer, 0.2 mM dNTPs, 2 U of Taq polymerase) according to the manufacturer’s recommendations and 3′ end amplification using specific 3′ primers and the universal reverse primer on an Eppendorf Mastercycler gradient PCR machine with the following cycling parameters: 10 cycles of touch-down PCR (denaturation: 94 °C for 30s; annealing: 55–68 °C for 30 s; extension: 72 °C for 30 s), followed by 30 cycles of regular PCR with annealing at 52 °C. Specific 5′ and 3′ primers were defined using KASpOD software.³⁴ (link)