Abstract
To date, there is no an established protocol for total RNA isolation in Egyptian buffalo
spermatozoa. The present study aimed (I) to establish a defined protocol for
total RNA isolation from fresh and frozen spermatozoa, (II) to evaluate RNA quality
and quantity from different extraction methods and studying gene expression.
Warm and standard room temperature modified QIAzol Lysis Reagents were used
for total RNA extraction. The quality and quantity of extracted RNA were checked,
and subsequently qRT-PCR was performed using androgen receptor-like and three
reference gene primers (GAPDH, ACTB and 18S). The warm modified QIAzol Lysis
Reagents resulted higher yield of good quality RNA from fresh (569.54 ± 18.83 ng/μl)
and frozen spermatozoa (110.59 ± 4.43 ng/μl), compared to standard room temperature
modified QIAzol (421.26 ± 7.18 ng/μl) and (29.07 ± 5.25 ng/μl), for fresh and frozen
semen samples respectively. The 260/280 ratio was 1.90 and 1.89 for fresh and
frozen isolated semen by warm method respectively. The integrity of RNA was good
and appeared as a sharp band on 2% agarose gel. The most stable reference gene was
18S. Reliable extraction method of high quality RNA yield could be a step forward for
understanding mechanisms of spermatogenesis for improving male fertility.