Applied Biosystems™ TaqMan™ Copy Number Assay

We have predesigned TaqMan Copy number assays for human and mouse, and we also have the Geneassist Copy number assay workflow builder tool that you may use to submit sequences for custom designed assays. Copy number variation experiments require a reference assay run in duplex with the target assay. We offer RNase P and TERT reference assays for human, and we offer Tfrc and TERT reference assays for mouse. The copy number assays are FAM-MGB labeled and the copy number reference assays are VIC-TAMRA labeled.

TaqMan Copy Number Assays are designed for analysis of copy number variations (CNVs) and smaller regions in human and mouse genomes. We have pre-designed assays for human and mouse genomes, and we also provide custom CNV assay design service for other species as well. A TaqMan Gene Copy Number Assay consists of two primers and a FAM-MGB probe in a 20X formulation. Like gene expression assays, measurements are made in real time. However, gene copy number assays use genomic DNA as the template and are run as a duplex reaction with a VIC-TAMRA (non-primer limited) labeled reference assay, which normalize the gDNA input for every reaction.

Copy Number Assay analysis has not been tested on, and is not available for OpenArray technology.

No, we do not. The TaqMan Copy Number Reference Assay, mouse, Tert (Cat. No. 4458368, 4458369) will not work either.

We have tested 10 µl on a 384 well plate for TaqMan CNV assays. We have not tested other volumes and do not recommend changing the protocol. You will need to empirically test any adjustments made to the CNV assay protocol.

We do not provide Copy Number Variation (CNV) assays to mtDNA. Cells contain high mtDNA copies (hundreds to thousands per cell). Our CNV Assay protocol is designed to analyze CNV changes in the 0-5 copy number range and is not appropriate for use with extremely high copy number differences. A gene expression–style relative quantitation approach might be best in this case.

If your data is not showing up in the CopyCaller Software, please confirm that the box is checked under the Assay Selection area on the right-hand side of the CopyCaller software. The box is in the left-most column under Assay Selection. This box must be selected for your graphical data to appear.

Please review the following possible causes and solutions:
-The reference sequence is not present or contains polymorphisms in the gDNA sample. Use an alternate copy number reference assay.
- There is variability in the amount of gDNA sample added to each reaction. Quantify the gDNA, then adjust the concentration of gDNA as needed.
Note: In general, the calculation of sample-level ΔCt accounts for variability in sample concentration.
- Pipetting was inaccurate. Check the pipette calibration of the pipettes, and pipette at least 5 µL of sample to prepare the reaction mix.

Most likely incorrect dyes were selected for each target. Check the dyes selected for each target, then reanalyze the data.

Most likely the sample evaporated. We recommend that you check the seal of the adhesive film for leaks before running the plate on the real-time PCR instrument.

Please review the following possible causes and solutions:
- There was insufficient Master Mix added to the reaction. Please follow accurate pipetting practices.
- Reagents are degraded. Ensure that the kits and reagents have been stored according to the instructions on the packaging and that they have not expired.

Please review the following possible causes and solutions:
-The sample evaporated. Check the seal of the adhesive film for leaks.
- The well is empty because of inaccurate pipetting. Check the calibration of the pipettes, and pipette at least 5 µL of sample.
- The well is assigned a sample or target in the plate document or experiment, but the well is empty. Ensure that the plate document or experiment is set up correctly. Then try excluding the well and reanalyzing the data.

Please review the following possible causes and solutions:
- The reagents were not mixed properly. Increase the length of time that you mix the reagents. We recommend that you confirm your mixing process by running a replicate assay.
- Pipetting was inaccurate. We recommend that you check the pipette calibration, and pipet at least 5 µL of sample to prepare the reaction mix.
- The threshold was not set correctly. Set the threshold above the noise level and where the replicates are tightest. See your real‑time PCR system user documentation for more information about setting the threshold.
-The concentration of one or more reaction components was incorrect. Ensure that the correct amounts of all the reactions components were added to the reaction plate.
- There was a low concentration of the target of interest. Rerun the assay with more gDNA template.
- Too much gDNA was added. We recommend that you reduce the amound of gDNA template added to the reaction mixture.
- The gDNA template or the amplicon is contaminated. Re-extract the gDNA and repeat the assay. Follow established PCR good laboratory practices to prevent contamination.

Please review the following possible causes and solutions:
- The reagents are contaminated with gDNA, amplicon or plasimd clones. We recommend that you esnure that your workspacea dn equpiment are cleaned correctly, then rerun the assay using new reagents. We also rsuggest that you use a Master Mix that contains UNG, such as TaqPath ProAmp Master Mix.
- The template or amplicon is contaminated. Please follow established PCR good laboratory practices to prevent contamination.

Please review the following possible causes and solutions:
- The copy number sequence is not present in the sample. First, run a sample with a known copy number for the target genomic region. Ensure that the Ct values for the reference assay (VIC dye) are normal in the samples without the copy number assay (FAM dye) signal. You can confirm your results by rerunning the sample using the same assay and/or running the sample using an alternative assay in the same genomic region, if available.

- There is a mismatch between the assay and the target sequence. We recommend that you perform bioinformatics for custom assays.
For example, ensure that the sequence submitted to assay design contains the correct target sequence. If needed, select an alternative target region for assay design or select another assay from the same genomic region. We also recommend using Custom Plus assays, which include bioinformatics.

- One or more of the PCR reaction components was not added. Check your pipetting equipment and/or technique.

- Incorrect dyes were selected for each target. Check the dyes selected for each target, then reanalyze the data.

- The annealing temperature was too high for the primers and/or probes. Ensure that the thermal cycler is set to the correct annealing and extension temperatures.
- The template is degraded. Determine the quality of the template, then rerun the assay with a fresh template if needed. We recommend that you use nuclease-free water.

- Inhibitors are present in the reaction. To check for inhibitors, we recommend that you run a serial dilution of your sample with an assay known to detect a tarfet in the sample (e.g. a reference assay). If an inhibitor is present, the high concentration samples yield higher-than-expected Ct values because the sample are not diluted.

- The baseline and/or threshold was improperly set. See your real-time PCR system user guide for procedures on setting the baseline and threshold. We recommend switching from an automatic baseline to a manual baseline (or vice versa), and/or lowering the threshold value to fall within the appropriate range.

Please review the following possible causes and solutions:
- The baseline was set improperly. We recommend switching from an automatic baseline to a manual baseline (or vice versa), and/or increasing the upper or lower value of the baseline range.
Please see your real‑time PCR system user guide for procedures on setting the baseline.
- The gDNA quality was poor. We recommend that you perform a quality check on the gDNA, then re-extract the gDNA if needed.
- There were difference concentrations caused my imprecise pipetting. Please follow accurate pipetting practices.
- The reagents or equipment are contaminated. Please ensure that your workspace and equipment are cleaned properly.

Please review the following possible causes and solutions:
- There is a sequence mismatch between the target region and the copy number assays. We recommend that you perform bioinformatics analysis, or select another assy from the same genomic region, if available.
- The reagents or the assays are degraded. Ensure that the kits and reagents have been stored according to the instructions on the packaging and that they have not expired. We recommend that you dilute the 60x assay to a 20x working stock to avoid excessive freeze-thaw cycles.
- The gDNA template is degraded or contaminated. Follow established PCR good laboratory practices.
- Inhibitors are present in the reaction. To confirm the presence of an inhibitor, we recommend that you run a serial dilution of your sample with an assay known to detect a target in the sample (e.g. a reference assay). If an inhibitor is present, low concentrations yield higher-than-expected Ct values because the sample is not diluted.

There may be interaction between the primer and probe. We recommend that you adjust the threshold manually, or select another assay from the same gene, if available. Alternatively, there may be bubbles in a well. In this case, we recommend that you repeat the assay with new reagents and centrifuge the reaction plate to ensure that it does not contain bubbles.

Please review the following possible causes and solutions:
- The baseline was set too high and some samples have Ct values lower than the baseline stop value. We recommend that you switch from manual to automatic baselining, or move the baseline stop value to a lower Ct. The baseline stop value should be set to a Ct 2 cycles before the amplification curve crosses the threshold. See your real‑time PCR system user guide for procedures on setting the baseline.
-No baseline can be set because the amplification signal is detected too early in the PCR cycles. We recommend that you use a gDNA concentration within the recommended range, and dilute the sample to increase the Ct value if needed.

A small ΔRn can mean that the PCR efficiency was poor. Ensure that the reagents were used at the correct concentration. Alternatively, the quantity of gDNA may be low (a low copy number of the target). In this case, we recommend that you increase the quantity of the gDNA in the reaction.

Most likely the fluorescence did not stabilize to the buffer conditions of the reaction mix.
Note: This condition does not affect PCR or the final results.
We recommend that you reset the lower value of the baseline range or use an automatic baseline.

Most likely the ROX dye was not set as the passive reference. Set ROX dye as the passive reference, then reanalyze the data.

Most likely the incorrect dye is set as the passive reference. Change the passive reference to the correct dye, then reanlyze the data.

When automatic baseline is used, the software selects baseline cycle values that are too narrow.
We recommend that you try the following:
-Change the analysis setting to allow manual adjustment, then set the Baseline Start Cycle to 3 and the Baseline End Cycle to 15.
- Set the Amplification Plot's graph type to linear. If amplification appears to begin earlier than cycle 15, adjust the Baseline End Cycle to 2 cycles prior to the start of amplification.
For example, if the amplification appears to begin at cycle 13, set the Start Cycle to 3 and End Cycle to 11.