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When compared to spherical conceptuses there was almost a 12-fold increase in mRNA expression for OSU-T1-50 in tubular conceptuses while expression in filamentous conceptuses increased over 100-fold.
Total RNA was extracted from individual conceptuses and pools of conceptuses of identical morphologies as previously described in our laboratory [ 8 ] . Briefly, conceptuses were denatured for 15 min on ice using 500 μl of denaturing solution (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 M 2-β-mercaptoethanol), 500 μl phenol, 70 μl 2 M sodium acetate (pH 4.0), and 140 μl chloroform/iso-amyl-alcohol (49:1 fresh dilution).
Quantitative analysis of clones of interest, specifically, s-adenosylhomocysteine hydrolase (SAHH), heat shock cognate 70 KD protein (HSC70), and one unknown transcript, OSU-T1-50, were assayed using quantitative real-time RT-PCR [ 14 ] . Individual and small pools of conceptuses were evaluated at the four morphologically distinct stages; spherical (n = 8), ovoid (n = 2), tubular (n = 5) and early filamentous (n = 6).
Gene expression of SAHH was similar between spherical, ovoid and tubular conceptuses, but there was nearly a 7-fold increase in gene expression in filamentous conceptuses.
Conceptuses were flushed into a sterile petri dish from the uterine horn and evaluated to determine an appropriate time-delayed removal of the second horn corresponding to the predicted time conceptuses would be in a tubular morphology as described by Geisert et al.
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