Critical steps

Step 31 Dilution and pipetting of cDNA are the most critical steps in the analysis and will introduce the greatest amount of error if done incorrectly. Always make fresh dilutions of cDNA on the day of the analysis and discard any diluted cDNA that remains. Do not freeze and reanalyze the diluted cDNA. Always dilute 50% more cDNA than you think you will need for that day. The original, undiluted cDNA is very stable and may be re-used for many years if stored properly.

Step 35 Apply the technique of 'pipette overloading' to deliver the correct amount of cDNA and reduce the aspiration and adhering of sample to the pipette tip. Depress the plunger of the multichannel pipette set for 5 ^l until it stops, then depress the plunger a little bit further. Place the pipette into the PCR strip tubes containing the dilute cDNA and pick up the sample. Add the 5 ^l sample to the side of the PCR plate by depressing the plunger until it stops. This will leave some residual solution in each tip. Discard the tips and repeat using new tips.

Step 38 Proper validation of the internal control gene is critical. The internal control gene should not change under experimental conditions (e.g. treated versus untreated or normal versus diseased tissue). Validation of the internal control should be done prior to quantifying the gene(s) of interest. Methodology to validate internal control genes has been described previously (Schmittgen et al., 2000). In brief, triplicate PCRs should be performed on each sample of cDNA (both treated and untreated samples) using primers for the internal control gene. Calculate the mean Ct for each cDNA and present the expression of the internal control as 2-Ct. Perform statistical analyses as described (Schmittgen et al., 2000) or use the students t-test to determine if the internal control gene has fluctuated under the conditions of the experiment.

Step 39 PCR efficiency is determined from the equation N = N0 x En, where N is the number of amplified molecules, N0 is the initial number of molecules, n is the number of PCR cycles and E is the efficiency which is ideally 2 (Mygind et al., 2002). When the equation is of the form n = -(1/log E) x log N0 + (log N/log E), a plot of log copy number versus Ct yields a straight line with a slope = -(1/log E) (Figure 2). Acceptable amplification efficiencies using this method are 1.85 to 2.05. Determination of amplification efficiency using this method has been described previously (Schmittgen et al., 2004).

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