Real-Time Quantitative RT-PCR
Total RNA was extracted from cultured cells with Tri Reagent (Sigma). Reverse transcription was carried out with an AMV reverse transcriptase kit (Invitrogen) on an Eppendorf Mastercycler (Eppendorf, Westbury, NY). To perform quantitative real-time RT-PCR, a standard curve was created for 500 pg to 250 ng cDNA using the Roche Light Cycler (Roche, Indianapolis, IN), with monitoring of the increasing fluorescence of the PCR products during amplification.
Upon establishing the standard curve, quantitation of the unknowns was determined with the Roche Light Cycler and adjusted to the quantitative expression of the beta-actin gene (ACTB) from the corresponding unknowns. Melting curve analysis determined the specificity of the amplified products and the absence of primer-dimer formation. All of the products obtained yielded the correct melting temperatures. The following primers were synthesized and gel-purified at the Yale DNA Synthesis Laboratory, Critical Technologies: CSF1 sense primer 50-GCTCAGCCAGATGCAA-30 and antisense primer 50-GTCCAGGTGGTCATG-30; and MIF sense primer 50-GTAGCCACATGATTGG-30 and antisense primer 50-GTTATCTCT-GAAGCGC-30. The ACTB sense and antisense primers were 50-CGTAC-CACTGGCATCGTGAT-30 and 50-GTGTTGGCGTACAGGTCTTTG-30, respectively. The expected sizes of the fragments amplified for CSF1, MIF and ACTB were 207 bp, 191 bp, and 459 bp, respectively.
Comparisons of the control and the various treatment groups were performed using the Kruskal-Wallis ANOVA on Ranks test followed by the Student-Newman-Keuls post hoc test with P < 0.05 representing statistical significance.