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Total RNA Purification from Cultured Cells Using the ABI PRISM 6700 Automated Nucleic Acid Workstation and Total RNA Lysis Reagents

detects the presence of such inhibitors. Applied Biosystems total RNA lysis reagents and purification technology readily removes them.

For very sensitive assessment of PCR performance, measure CT values on fourfold serial dilutions of an individual sample. To measure inhibition, plot a standard curve of the three most dilute samples and extrapolate to compare measured versus calculated CT values for the 1:4 and undiluted sample. PCR inhibitor is present if the measured CT value for the undiluted sample is suppressed by >0.5 cycles from the calculated CT value (Figure 8).

Cross-contamination Intra/inter-run, or well-to-well: <1 in 106 copies at an occurrence rate of ≤1 per tray

Range of Recovery

Figure 6 shows that for Raji cells, total RNA recovery versus cell input is linear for the range 10 pg100 μg, (101106 cells).

Caution: Input ranges >106 cells/well can cause blockages in the purification tray, which, in turn, causes overflow of the wash solution. This results in well-to-well cross-contamination and failure of the purification run (Figure 9).


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