PCR Inhibition and Cell Input
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Kit Components
The TaqMan® Cells-to-CT Control Kit is designed for use with the TaqMan Gene Expression Cells-to-CT Kit, the TaqMan PreAmp Cells-to-CT Kit, and the TaqMan Fast Cells-to-CT Kit. The Control Kit includes Xeno RNA™ Control, a synthetic RNA transcript with a unique sequence that lacks homology to current annotated biological sequences, making it an ideal control for any experiment. The Control Kit also includes a TaqMan Gene Expression Assay for the Xeno RNA Control target and a TaqMan Gene Expression Assay (ACTB) for the highly expressed β-actin gene. β-actin functions as an endogenous control for normalization during relative quantitation calculations (ΔΔCT method). This TaqMan β-actin assay has been shown to detect β-actin from human, mouse, rat, monkey, hamster, and pig.
Control for Reaction Inhibitors and Lysis Efficiency
The Xeno RNA Control confirms that samples can support reverse transcription and real-time PCR (real-time RT-PCR) and is an indicator of reaction inhibitors. By adding the Xeno RNA Control to the Stop Solution during the last step of the cell lysis procedure, each sample is provided with a constant amount of control target that is subsequently amplified by the Xeno RNA TaqMan Gene Expression Assay. Availability of Xeno RNA for amplification is not affected by lysis efficiency, thus the CT for Xeno RNA should not change as cell input changes, in the absence of RT and PCR inhibitors (Figure 1, Panels A and B). Nonlinearity of the Xeno RNA Control plot indicates the presence of RT or PCR inhibitors at high cell numbers (Figure 1, Panel C).
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).
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
Amplification with the TaqMan β-actin Gene Expression Assay can be used to verify that the lysis reaction contains a sufficient number of cells, an important control for experiments with sample inputs of less than ~100 cells. If a target gene is expressed at a low level, it is difficult to distinguish between inadequate sample input for RT or PCR and an undetectable level of gene expression. In Figure 2, the plot of CT versus cell input for β-actin is linear, indicating efficient cell lysis at all cell concentrations and verifying the presence of cells in the 10-cell lysates. The linear plot of CT versus cell input for Xeno RNA Control indicates lack of RT-PCR inhibitors at the highest cell inputs. The CT plot for the experimental target indicates that the target transcript cannot be reliably detected below the 100-cell input level. Linear detection of the target at higher cell numbers confirms this conclusion.
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.
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
The TaqMan Cells-to-CT Control Kit is also ideal for assessing the compatibility of untested cell lines with the TaqMan Cells-to-CT Kits and for determining the maximum number of cells that can be used per lysis reaction. To do this, prepare serial dilutions of the cells being tested, then prepare cell lysates with the TaqMan Cells-to-CT Kit of choice, including the Xeno RNA Control. Use each lysate in parallel real-time RT-PCRs with the supplied Xeno RNA Control and β-actin TaqMan Gene Expression Assays. Depending on the experimental goals, it may also be useful to include a third set of TaqMan Gene Expression Assay PCRs that target the gene of interest, to determine the lower limit of cell input that still allows for reliable target detection.
Scientific Contributors
Laura Chapman, Annalee Nguyen, Quoc Hoang, Richard Fekete • Applied Biosystems, Austin, TX
Scientific Contributors
Laura Chapman, Annalee Nguyen, Quoc Hoang, Richard Fekete • Applied Biosystems, Austin, TX
