Optimized semiquantitative RT-PCR to study ebaf temporal expression pattern during the early pregnancy in the rat oviduct

MARTIN E. ARGAÑARAZ; PABLO A. VALDECANTOS; DORA C. MICELI

Pinamar, Buenos Aires

Congreso; XLI Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular - SAIB; 2005

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

In previous work we have cloned and identified the ebaf (endometrial bleeding-associated factor) gene complete CDS (Accession No. AY758558) that is specifically expressed in the rat oviduct. To asses ebaf mRNA levels in early stages of pregnant rats, we optimized a semiquantitative RT-PCR standard procedure. The protocol was modified taking into account some aspects of the Real Time PCR technique, trying to: a) minimize the multiple variables that affect a single PCR reaction b) maximize gene expression differences, working at the exponential phase of the reaction. First we determined the amplification linear ranges for both ebaf and B-actin, as housekeeping gene, assaying different cycle numbers to obtain the complete profile of the PCR reaction (10 to 40 cycles). Each sample was then amplified in 6 individual PCR reactions for ebaf (21 to 26 cycles), and for the B-actin gene (18 to 23 cycles). By this way we ensure a linear amplification efficiency and avoid the reaction variability. Three different samples for each pregnancy day were assayed (day 1 to 7) and gene expression was expressed as ebaf/ B-actin ratio. The experiment shows that ebaf expression was higher at day 4 of pregnancy compared to day 1 and 7 (five folds), suggesting a relevant function in the oviduct during the early stages of pregnancy.