Objective: To establish and optimize a real time RT-PCR system for determining the transcript levels of CatSper1 in human and mouse mature spermatozoa containing microamount of RNA.
Methods: Total RNA of human and mouse mature spermatozoa was isolated by using TRIzol reagent and reversely transcribed to complementary DNA respectively. Primers for real time RT-PCR were designed in the homologous area of the human and mouse CatSper1 mRNAs. Human sperm complementary DNA was used as the template to the optimize the conditions for SYBR Green I real time RT-PCR, including annealing temperature, Mg2+ concentration, fluorescence measurement temperature and the ratio between forward and reverse primers. The standard curve was constructed with serial dilutions of complementary DNA from human sperm to ascertain the amplification efficiency of SYBR Green I real time PCR and to quantitate the CatSper1 mRNA levels in the human and mouse mature spermatozoa.
Results: The optimal conditions for real time RT-PCR, that is, annealing temperature, Mg2+ concentration and the ratio between forward and reverse primers were 63 degrees C, 3.0 mmol/L and 1:1 respectively. The fluorescence measurement temperature was 88 degrees C. The standard curves were Y = -3.402 log (X) + 25.99 and Y = -3.409 log(X) + 24.09 in the human sperm cDNA and mouse sperm cDNA as the template, with amplification efficiency of 96.8% and 96.5% respectively. The R2 value (an indicator of the quality of the fit of the standard curve to the standard data points plotted) of both standard curves was 0.998. The CatSper1 mRNA levels in the human and mouse mature spermatozoa could be determined according to the standard curve.
Conclusion: The general RT-PCR system, by adding SYBR Green I and optimizing its conditions, could be used to quantitate the mRNA levels in both human and mouse mature spermatozoa.