PCR Inhibition and Cell Input
Control for Reaction Inhibitors and Lysis Efficiency
Figure 1. Illustration of Pilot Experiment to Determine Maximum Number of Cells per Lysis Reaction. By adding Xeno RNA™ Control to the Stop Solution used to prepare TaqMan® Gene Expression Cells-to-CT™ Kit lysates, the Xeno RNA TaqMan Gene Expression Assay can be used to evaluate increasing amounts of cells per lysis reaction for inhibitors of real-time RT-PCR. In parallel, the cDNA from these same lysates can be amplified using the β-actin TaqMan Gene Expression Assay (ACTB) to verify that sample lysis is complete and that cellular RNA is available for RT-PCR. Panel A: Linearity for both Xeno RNA Control and β-actin assays indicates no RT-PCR inhibitors and efficient lysis at all cell inputs. Panel B: Linearity for Xeno RNA Control indicates no RT-PCR inhibitors, but non-linearity for b-actin indicates incomplete lysis at higher cell inputs. Panel C: Non-linearity for Xeno RNA Control indicates the presence of RT-PCR inhibitors at higher cell inputs. Because of inhibitors, lysis efficiency at higher cell inputs is unknown. Data for illustration purposes only. Dashed lines in Panels B and C represent linearity expected without inhibitors and with efficient lysis at all cell inputs.
The assay for the endogenous gene control β-actin allows monitoring of lysis efficiency: CT should decrease in a linear fashion as cell numbers increase (Figure 1, Panel A). Deviation from linearity as cell input increases, while the Xeno RNA CT plot remains linear, indicates incomplete lysis at high cell numbers (Figure 1, Panel B).
Control for Sufficient Cell Number
Figure 2. Illustration of Control Experiment to Verify Sufficient Cell Input. Xeno RNA™ TaqMan® Gene Expression Assay is used to detect inhibitors of real-time RT-PCR as cell numbers per lysis reaction are increased, as described in Figure 1. The β-actin TaqMan Gene Expression Assay verifies that sample lysis is adequate at all levels of cell input and that cellular RNA is available for RT-PCR. The CT plot for the experimental target indicates that expression of this gene does not allow reliable detection at the lowest cell input level. Data for illustration purposes only.
Assess Compatibility of Untested Cell Lines
Laura Chapman, Annalee Nguyen, Quoc Hoang, Richard Fekete • Applied Biosystems, Austin, TX