Changes in Uterine Expression of Leukemia: MATERIALS AND METHODS(4)

RNA Isolation and cDNA Preparation

Total RNA was isolated from the uterine cornua of individual animals by homogenization in guanidium thiocy-anate followed by centrifugation through a cesium chloride gradient. RNA was quantified using A260/A280 spec-trophotometric readings. Total RNA (1 ^g) from skunk uteri was prepared for reverse transcription-polymerase chain reaction (RT-PCR) by treatment with DNase. After addition of 50 ng random hexamers, samples were heat denatured at 75°C for 5 min and transferred immediately to ice. RT reactions were carried out as described previously. Identical uterine RNA samples were incubated in parallel but without reverse transcriptase to verify that all endogenous genomic DNA had been eliminated. Cheap Diskus Advair

A 1-^l aliquot of the resultant cDNA was mixed with specific primers and subjected to PCR (single-strength buffer, 0.2 mM deoxyribonucleotides, 3 mM MgCl2, 0.4 ^M each primer, 1 U Taq polymerase) in order to amplify skunk cyclophilin and LIF sequences. PCR amplification of cyclophilin yielded a 195-base pair (bp) fragment while the LIF primers amplified a 483-bp fragment. Products were separated by electrophoresis, cloned, and identified by sequencing in both directions. Homologous primers that were internal to the cloning sites for LIF and that spanned an intron were selected for use in the quantitative RT-PCR assay and yielded a 398-bp PCR product from cDNA templates.

This entry was posted in Leukemia and tagged Inhibitory Factor, Leukemia, Pregnancy.