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  • Comparative Studies on the In Vitro: MATERIALS AND METHODS(6)

The H3.3 PCR product was used as the internal standard for all samples. In all tissues, H3.3 is a histone that is constitutively expressed and that does not change over time or with hormone treatment. buy asthma inhalers

RT was performed in a final volume of 20 ^l with 1 ^g RNA and 50 U/^l murine leukemia virus reverse transcriptase using the GeneAmp RNA PCR core kit (Perkin-Elmer, Irvine, CA). The mixture was incubated at 42°C for 30 min. The RT product was aliquoted equally into two tubes and primers were added (50 pmol/tube); the volume was adjusted to 40 ^l, and PCR amplification was performed with 0.5 ^l of 5 U/ml Taq polymerase (Perkin-Elmer) and 0.25 ^l of 10 mCi/ml [32P]dCTP per tube. Amplification was allowed to occur for 24 cycles consisting of 94°C (1 min), 60°C (2 min), and 72°C (3 min) followed by 15 min of final extension at 72°C. Primers for H3.3, IGFBP-1, and aSMA were added together in a single tube so that amplification of the cDNAs for H3.3, IGFBP-1, and aSMA occurred in the same tube. Also, RIa, RIp, RIIa, and RIIp cDNAs were amplified together in a single tube. A linear curve was plotted using number of cycles of amplification versus densi-tometric values of the PCR products, measured with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) (see Fig. 4C). The optimal number of cycles for amplification that fit within the linear range was chosen for each set of primers.
The PCR products were electrophoresed in 1.5% agarose gels, and the gels were dried and exposed to film. Data were quantified by image analysis using the Phosphor-Imager.

Statistical Analysis

Data were analyzed by least-squares ANOVA using the SuperANOVA software package (Abacus Concepts, Inc., Berkeley, CA). Sources of variation included experiment (defined as tissues from different animals/women), treatment (presence or absence of hormones/dbcAMP), days of treatment, and their interactions. For short-term dbcAMP treatment experiments, orthogonal contrasts were used to compare the means of the various treatments (see Fig. 5).

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