DRAFT
September 1, 2004 11:39 am, CH_Trouble.fm
Low Precision or Irreproducibility
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 8-5
Low Precision or Irreproducibility
Overview
There are many reasons why an assay run with the Applied Biosystems 7900HT Fast
Real-Time PCR System can have less than optimal precision. Factors that can affect
precision are described in detail below.
Improper Threshold Setting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-5
Imprecise Pipetting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-6
Non-Optimized Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-6
Incomplete Mixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-6
Air Bubbles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-6
Splashing PCR Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-6
Drops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-7
Writing on the Reaction Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-7
Fluorescent Contamination on the Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-7
Errors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-7
Contaminated Sample Block. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-7
Improper or Damaged Plastics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-8
Low Copy Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-8
Use of Non-Applied Biosystems PCR Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . 8-8
Improper
Threshold Setting
The key to high-precision quantitative PCR is accurate detection of the geometric
phase. The Applied Biosystems 7900HT Fast Real-Time PCR System typically
delivers sufficient sensitivity so that at least three cycles of the geometric phase are
visible, assuming reasonably optimized PCR conditions. The SDS software
calculates a fixed signal intensity, called a threshold, that each signal generated from
PCR amplification must reach before it is recognized by the software as actual
amplification. The calculated threshold is an approximation, and should be examined
and modified as needed.
Modifying the Threshold
In a real-time document of the SDS software, the threshold can be modified via the
Amplification plot view following analysis of the run data. See “Setting the Baseline
and Threshold Values” on page 6-46 for more information.
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