Table 11.3(Continued) To Enhance this Manipulate One or More of These Components Use appropriate controls. Repeat experiments when data are outside f standard deviation limits Minimize the manipulations from start to Check that the temperature profile is consistent at every position in the heating Control Confirm that controls have similar sequence rofile and amplification efficien Confirm that PCR was linear by producing Analysis Use an automated system to reduce handling steps Detection Check the detection strategys senitivity and ability to measure vield in the al phase of PCR Confirm that the technique has high High-throughput Instrument Select a system that handles microtiter plates and multiple samp multaneously Reaction Use a hot-start PCR strategy(d'aquilla et al., 1991; Chous et al., 1992: Kellogg Use a master PCR reagent mix me aterial: dont different lots. Sample preparation Use of robotics. Storage of sample as cDNA or ethanol precipitate, rather than RNA in solution. Cycling Use one cycling strategy for all samples. Analysis Use an automated system. Detection Use an automated detection system to monitor the exponential phase. Detection Monitor specific PCR product formation by hybridization via nucleic acid probe. Use fluorescent intercalating dye nested PCR t al., 1990). Note: Sensitivity is gained at the expense of quantitation. Use a hot-start PCR strategy Use UNG and dUTp to prevent carryover Analysis Use a real time PCR strategy that detects low levels of amplicon missed by gel PCR 299PCR 299 Table 11.3 (Continued) To Enhance This Parameter Manipulate One or More of These Components Use appropriate controls. Repeat experiments when data are outside of standard deviation limits. Minimize the manipulations from start to finish. Cycler Check that the temperature profile is consistent at every position in the heating block. Control Confirm that controls have similar sequence profile and amplification efficiency. Confirm that PCR was linear by producing a standard curve. Analysis Use an automated system to reduce handling steps. Detection Check the detection strategy’s senitivity and ability to measure yield in the exponential phase of PCR. Confirm that the technique has high sensitivity and magnitude over a wide dynamic range. High-throughput Instrument Select a system that handles microtiter plates and multiple sample simultaneously. Reaction Use a hot-start PCR strategy (D’Aquilla et al., 1991; Chous et al., 1992; Kellogg et al., 1994). Use a master PCR reagent mix. Use aliquots taken from the same lot of material; don’t mix aliquots from different lots. Sample preparation Use of robotics. Storage of sample as cDNA or ethanol precipitate, rather than RNA in solution. Cycling Use one cycling strategy for all samples. Decrease the cycling time. Analysis Use an automated system. Detection Use an automated detection system to monitor the exponential phase. Sensitivity Detection Monitor specific PCR product formation by hybridization via nucleic acid probe. Use fluorescent intercalating dye (Wittwer et al., 1997). Reaction Use a nested PCR strategy (Simmonds et al., 1990). Note: Sensitivity is gained at the expense of quantitation. Use a hot-start PCR strategy. Use UNG and dUTP to prevent carryover. Analysis Use a real time PCR strategy that detects low levels of amplicon missed by gel