METHODS and MATERIALS: A Protocol for Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
2 m g of total RNA are converted to cDNA with the Superscript TM Preamplification Kit (Gibco BRL, Gaithersburg , MD ), following the manufacturer’s recommendations. The final volume is 20 m l. 2 PCR primers are utilized to amplify the cDNA, with Forward and Reverse Primers. Actin primers are also amplified as controls. PCR is performed in a 50 m l reaction mixture, containing 1 m l of cDNA, 10 mmol/L Tris-HCl buffer (pH 8.3), 50 mmol/L of KCl, 200 m M dNTPs (deoxynucleoside triphosphates), 100 ng primers and 2.5 units of HotstarTaq TM DNA polymerase (QIAGEN) on a Thermal Cycler. In all PCR reactions, a blank control was included with water as the template. The same PCR conditions are utilized for both a control gene, and the selected gene for amplification. Cycles are 95 o C for 15 minutes to activate the polymerase, followed by 30 cycles for of denaturing at 94 o C for 30 seconds, annealing at 60 o C for 30 seconds and extension at 72 o C for 30 seconds. The final extension is at 72 o C for 10 minutes. Equal amounts of PCR products are then separated on a 2.5% agarose gel, stained with ethidium bromide and subsequently visualized under ultraviolet (UV) light for visualization and comparisons.