2.8 RT-PCR

The expression of lamin A and progerin was assessed using a reverse transcription-polymerase chain reaction (RT-PCR) system to accurately determine the mRNA levels of lamin A, progerin and, as an internal control, beta-actin. The design of the primers for progerin and lamin A PCR took into account the splicing characteristics of the mRNAs used for the synthesis of the corresponding proteins–each pair of oligonucleotides amplified only its ‘own’ template (361 bp for lamin A, 222 bp for progerin). Total RNA from spermatozoa without significant pathology or spermatozoa from a patient with globozoospermia was isolated using the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s recommended protocol. The first strand of cDNA was synthesized using 100 ng of RNA per 20 μL of the reaction mixture, then a 1/10 aliquot was used as a template for PCR.