# Risk-Based QC Planning for Molecular Assays (PCR, NGS)

## Quick Answer

- Design QC for PCR and NGS around specific failure modes such as contamination, amplification bias, and base-calling errors instead of applying a universal control set.
- Select positive, negative, and internal controls based on the highest-risk steps in your workflow, then monitor results with statistical process rules.
- A risk-based QC plan cannot eliminate all errors, and validation data from one instrument or reagent lot may not transfer to another platform.

## At a Glance

| Risk Category | PCR Failure Mode | NGS Failure Mode | Recommended Control Layer |
|---|---|---|---|
| Pre-Analytical | Carryover contamination from amplicons or environmental nucleic acids | Adapter contamination or index hopping during library preparation | Negative controls, reagent blanks, and workspace segregation |
| Analytical | Primer dimer formation, amplification bias, or polymerase errors | Sequencing depth variation, base-call errors, or alignment artifacts | Positive controls with known concentrations and internal spike-in controls |
| Post-Analytical | Incorrect threshold cycle interpretation or poor melt-curve analysis | Variant calling errors or false-positive mutation detection | Statistical monitoring, replicate analysis, and orthogonal confirmation |

## Scope and Purpose of Risk-Based QC Planning

Molecular assays that use polymerase chain reaction (PCR) and next-generation sequencing (NGS) share a common goal: converting nucleic acid templates into interpretable data. The failure modes, however, differ substantially between the two platforms. PCR amplifies a target region and produces quantitative or qualitative signals, while NGS generates millions of sequence reads that require complex bioinformatics. A quality control (QC) plan that treats both platforms identically will miss critical failure points and may produce false confidence in results.

The purpose of a risk-based QC plan is to allocate control resources where they matter most. Instead of running every possible control on every sample, you identify the steps where errors are most likely to occur, where errors would have the greatest impact on the final result, and where controls can actually detect those errors. This approach follows the same logic used in biosafety risk assessment, where the level of containment and oversight is matched to the hazard and the procedure being performed. The CDC and NIH describe this principle in the Biosafety in Microbiological and Biomedical Laboratories, where risk assessment considers the agent, the procedure, and the laboratory environment before selecting controls.

For molecular assays, the equivalent process involves mapping each step from sample collection to final data interpretation, then assigning a risk level to each step. High-risk steps receive more controls and more frequent monitoring. Low-risk steps receive minimal controls. This does not mean that low-risk steps are ignored. It means that the QC burden is proportional to the probability and consequence of failure.

The National Library of Medicine provides a broad gateway to molecular biology and laboratory methods that can help you understand the technical basis of each step. The MIQE guidelines, published in Clinical Chemistry, provide a detailed framework for qPCR reporting and control selection. These guidelines emphasize transparency in reporting and the need to document the conditions under which an assay performs correctly.

A risk-based QC plan is not a static document. It changes when you introduce a new reagent lot, a new instrument, a new operator, or a new sample type. Each change introduces new variables that can shift the risk profile. The plan should be reviewed at defined intervals and after any significant failure.

## Core Principles of Risk-Based QC

### Risk Identification

Risk identification begins with a complete workflow map. For PCR, the workflow includes sample collection, nucleic acid extraction, reverse transcription if RNA is the target, primer design, amplification, and detection. For NGS, the workflow includes all of the PCR steps plus library preparation, adapter ligation, indexing, clonal amplification, sequencing, and bioinformatic analysis.

Each step has a distinct set of failure modes. Sample collection can introduce inhibitors or degrade the target. Nucleic acid extraction can lose material or introduce contaminants. Reverse transcription can fail to convert all RNA to cDNA. Amplification can produce primer dimers or bias certain sequences. Library preparation can introduce adapter dimers or index misassignment. Sequencing can produce base-call errors. Data analysis can misalign reads or call false variants.

The risk identification step should be performed by the people who actually run the assay. They know where the process is fragile and where the controls are already working. The output of this step is a list of failure modes with a preliminary risk rating.

### Risk Analysis and Evaluation

Risk analysis assigns a probability and a consequence to each failure mode. Probability is the likelihood that the failure occurs under normal operating conditions. Consequence is the impact of the failure on the final result. A failure that occurs rarely but produces a completely wrong clinical result is high risk. A failure that occurs frequently but is easily detected and corrected is lower risk.

The risk rating should be based on data where possible. If you have run the assay for a year, you can estimate the frequency of contamination events from your negative control records. If you are introducing a new assay, you can use the validation data to estimate the failure rate. The MIQE guidelines emphasize the importance of documenting the technical parameters of an assay, including the efficiency, the limit of detection, and the precision. These parameters provide the baseline for risk analysis.

Risk analysis should also consider the cost of a failure. A false-negative result in a diagnostic assay may lead to a missed diagnosis. A false-positive result may lead to unnecessary treatment. In a research setting, a false result may lead to a wasted experiment or an incorrect conclusion. The cost of failure is not always financial. It can be scientific, clinical, or regulatory.

### Risk Evaluation and Control Selection

Risk evaluation is the step where you decide which risks are acceptable and which require additional controls. The controls available for PCR and NGS include:

- Positive controls: a known template that should produce a known result
- Negative controls: a sample that should produce no signal
- Internal controls: a template that is added to every sample to monitor extraction, amplification, or sequencing efficiency
- Replicate controls: the same sample run multiple times to assess precision
- Calibration controls: a series of known concentrations used to establish a standard curve

The selection of controls should be driven by the risk analysis. If contamination is the highest risk, then negative controls should be placed throughout the workflow. If amplification efficiency is the highest risk, then a standard curve should be run with each batch. If the risk is in the bioinformatics step, then a known variant should be spiked into the data analysis pipeline.

The MIQE guidelines provide a detailed list of parameters that should be reported for qPCR assays. These include the number of technical replicates, the number of biological replicates, the efficiency of the reaction, the limit of detection, and the limit of quantification. The guidelines also recommend that the assay design be described in enough detail that another laboratory could reproduce it.

## Practical Workflow for Building a Risk-Based QC Plan

### Step 1: Define the Assay and Its Intended Use

The first step is to define the assay and its intended use. Is the assay qualitative or quantitative? Is it for research or for clinical diagnosis? What is the sample type? What is the target sequence? What is the expected range of target concentrations?

The intended use determines the level of rigor required. A research assay that is used to screen for the presence of a gene may require fewer controls than a clinical assay that is used to determine the viral load of a patient. The MIQE guidelines provide a framework for reporting the technical parameters of a qPCR assay, and these parameters should be defined before the QC plan is built.

### Step 2: Map the Workflow and Identify Failure Points

Draw a flow diagram of the entire workflow, from sample collection to final data interpretation. For each step, list the possible failure modes. For example, in the extraction step, the failure modes might include: incomplete lysis, degradation of the nucleic acid, carryover of inhibitors, and contamination from the extraction reagents.

The Addgene Protocols provide educational context for common molecular biology workflows, including the preparation of reagents and the handling of samples. These protocols are not universally validated, but they can help you identify the steps where errors are likely to occur.

### Step 3: Assign Risk Ratings

For each failure mode, assign a probability and a consequence rating. Use a simple scale, such as low, medium, or high. The probability rating should be based on your experience with the assay or on the published data. The consequence rating should be based on the impact of the failure on the final result.

The risk rating should be documented in a table or a spreadsheet. This table becomes the basis for the QC plan. It should be reviewed and updated whenever the assay is changed.

### Step 4: Select Controls and Monitoring Strategies

Based on the risk ratings, select the controls that will be used. The controls should be placed at the steps where the risk is highest. For example, if the risk of contamination is high, then negative controls should be placed at the extraction step, the amplification step, and the detection step. If the risk of amplification failure is high, then a positive control should be included in every run.

The monitoring strategy should also be defined. How often will the controls be run? What are the acceptance criteria? What action will be taken if a control fails? The acceptance criteria should be based on the performance of the assay, not on an arbitrary threshold.

### Step 5: Document the Plan and Train the Staff

The QC plan should be documented in a standard operating procedure. The document should include the workflow diagram, the risk table, the control selection, the acceptance criteria, and the corrective actions. The staff should be trained on the plan and should be able to explain the rationale for each control.

The documentation should also include the records that will be kept. The records should include the results of the controls, the lot numbers of the reagents, the instrument used, and the operator. These records are essential for troubleshooting and for demonstrating that the QC plan is being followed.

### Step 6: Review and Update the Plan

The plan should be reviewed at regular intervals, such as every six months or every year. The review should include an analysis of the control records. If the controls are failing more often than expected, the risk ratings should be updated and the controls should be adjusted. If the controls are never failing, the plan may be over-controlled and some controls can be removed.

The review should also be triggered by any significant change to the assay, such as a new reagent lot, a new instrument, or a new operator.

## Selecting Controls for PCR Assays

### Positive Controls

A positive control is a known template that should produce a known result. The positive control should be designed to mimic the target as closely as possible. For a qualitative assay, the positive control should be a template that is known to be present in the sample. For a quantitative assay, the positive control should be a template with a known concentration.

The positive control should be run in every assay. The result of the positive control should be within the acceptance criteria. If the positive control fails, the assay is invalid and the results should not be reported.

The positive control can also be used to monitor the efficiency of the amplification. If the positive control is a standard curve, the slope of the curve can be used to calculate the efficiency. The efficiency should be within the range specified by the assay design.

### Negative Controls

The negative control is a control that should produce no signal. The negative control is used to detect contamination. The negative control should be placed at the steps where contamination is most likely to occur. For example, a negative control should be placed at the extraction step to detect contamination of the extraction reagents. A negative control should be placed at the amplification step to detect contamination of the amplification reagents.

The negative control should be a sample that is known to be free of the target. The negative control can be water, a buffer, or a sample that is known to be negative. The negative control should be treated in the same way as the sample, including the extraction and the amplification steps.

If the negative control produces a signal, the assay is invalid. The source of the contamination should be identified and eliminated before the assay is repeated.

### Internal Controls

The internal control is a control that is added to every sample. The internal control is used to monitor the efficiency of the extraction and the amplification. The internal control is a template that is different from the target. The internal control is amplified with a different set of primers.

The internal control can be a synthetic template or a housekeeping gene. The internal control should be added to the sample before the extraction step. The internal control should be amplified in the same reaction as the target.

The result of the internal control is used to determine if the sample was extracted and amplified correctly. If the internal control fails, the sample is invalid and the result should not be reported.

The internal control is especially important for clinical samples, where the sample may contain inhibitors that prevent the amplification. The internal control can detect the presence of inhibitors.

## Statistical Monitoring of QC Data

### Levey-Jennings Charts

The Levey-Jennings chart is a plot of the control results over time. The chart is used to detect trends and shifts in the assay performance. The chart is a plot of the control result on the y-axis and the time on the x-axis. The chart includes the mean and the standard deviation of the control results.

The Levey-Jennings chart is used to detect the following patterns:

- A single point outside the 3 standard deviation limit
- Two consecutive points outside the 2 standard deviation limit
- A run of 10 points on one side of the mean
- A trend of increasing or decreasing values

The Levey-Jennings chart is a simple and effective tool for monitoring the assay performance. The chart should be reviewed after each run. If the chart shows a pattern, the assay should be investigated.

### Westgard Rules

The Westgard rules are a set of rules for interpreting the control results. The rules are used to determine if the assay is in control or out of control. The rules are based on the mean and the standard deviation of the control results.

The Westgard rules include:

- 1-3s: one control result exceeds the mean plus or minus 3 standard deviations
- 2-2s: two consecutive control results exceed the mean plus or minus 2 standard deviations
- R-4s: one control result exceeds the mean plus 2 standard deviations and another control result exceeds the mean minus 2 standard deviations
- 4-1s: four consecutive control results exceed the mean plus or minus 1 standard deviation
- 10x: ten consecutive control results are on one side of the mean

The Westgard rules are used to determine if the assay is in control. If any of the rules are violated, the assay is out of control and the results should not be reported.

The Westgard rules are a valuable tool for the statistical monitoring of the assay. The rules are used to detect the errors that are not visible in the individual control results.

### The Role of NIST in Measurement Quality

The National Institute of Standards and Technology (NIST) provides the measurement science that is the basis for the quality of the laboratory results. NIST develops the standard reference materials and the calibration standards that are used to ensure the accuracy of the measurements. The NIST laboratory programs provide the framework for the measurement quality.

The NIST standards are used to calibrate the instruments and to validate the assays. The NIST standards are also used to ensure the traceability of the measurements. The traceability is the ability to relate the measurement result to a national standard.

The use of the NIST standards is important for the accuracy of the assay. The standards should be used to calibrate the instruments and to validate the assays. The standards should be used to ensure the accuracy of the results.

## Risk-Based QC for NGS Assays

### Library Preparation Controls

The library preparation is the step where the DNA is fragmented, the adapters are added, and the library is amplified. The library preparation is a high-risk step because the errors in this step can be amplified in the sequencing step.

The controls for the library preparation include:

- A positive control library that is known to produce a good result
- A negative control library that is known to produce no result
- An internal control that is added to every library

The positive control library is used to monitor the efficiency of the library preparation. The positive control library should be a library that is known to produce a good result. The positive control library should be prepared in the same way as the sample libraries.

The negative control library is used to detect contamination. The negative control library should be a library that is known to produce no result. The negative control library should be prepared in the same way as the sample libraries.

The internal control library is used to monitor the efficiency of the library preparation. The internal control library is a library that is added to every sample. The internal control library is a library that is designed to be different from the target.

### Sequencing Run Controls

The sequencing run controls are the controls that are used to monitor the sequencing run. The sequencing run controls include the control library and the control.

The control library is a library that is known to produce a good result. The control library is used to monitor the efficiency of the sequencing run. The control library is a library that is known to produce a good result.

The control is a control that is used to monitor the quality of the sequencing run. The control is a control that is used to monitor the quality of the sequencing run.

The sequencing run controls are used to determine if the sequencing run is successful. If the sequencing run controls fail, the sequencing run is invalid and the results should not be reported.

### Bioinformatics Controls

The bioinformatics controls are the controls that are used to monitor the data analysis. The bioinformatics controls include the control and the control.

The control is a control that is used to monitor the accuracy of the data analysis. The control is a control that is used to monitor the accuracy of the data analysis.

The control is a control that is used to monitor the accuracy of the data analysis. The control is a control that is used to monitor the accuracy of the data analysis.

The bioinformatics controls are used to determine if the data analysis is successful. If the bioinformatics controls fail, the data analysis is invalid and the results should not be reported.

## Common Failure Patterns and Troubleshooting

### Contamination

Contamination is the most common failure pattern in PCR and NGS. Contamination can be caused by the carryover of the amplicons, the contamination of the reagents, or the contamination of the environment.

The contamination is detected by the negative controls. If the negative controls produce a signal, the contamination is present. The contamination should be investigated and the source should be identified.

The contamination can be prevented by the use of the separate areas for the pre-amplification and the post-amplification steps. The contamination can be prevented by the use of the filtered pipette tips and the use of the clean benches.

### Amplification Failure

The amplification failure is the failure of the amplification to produce the expected product. The amplification failure can be caused by the inhibitors in the sample, the poor primer design, or the incorrect reaction conditions.

The amplification failure is detected by the positive controls. If the positive controls fail, the amplification is invalid. The amplification failure is investigated by the use of the internal controls.

The amplification failure can be prevented by the use of the internal controls and the use of the optimized reaction conditions.

### Base-Call Errors

The base-call errors are the errors in the sequencing data. The base-call errors can be caused by the sequencing instrument, the sequencing chemistry, or the data analysis.

The base-call errors are detected by the sequencing run controls. If the sequencing run controls fail, the sequencing run is invalid. The base-call errors are investigated by the use of the bioinformatics controls.

The base-call errors can be prevented by the use of the high-quality sequencing reagents and the correct data analysis.

## Biosafety and Regulatory Context

The risk-based QC plan should be integrated with the biosafety and the regulatory requirements. The biosafety requirements are described in the Biosafety in Microbiological and Biomedical Laboratories. The biosafety requirements include the use of the containment, the use of the personal protective equipment, and the use of the institutional oversight.

The NIH Guidelines for Recombinant or Synthetic Nucleic Acid Molecules provide the oversight for the research that involves the recombinant or synthetic nucleic acid molecules. The guidelines describe the requirements for the containment and the oversight of the research.

The risk-based QC plan should be integrated with the biosafety requirements. The QC plan should be designed to be compatible with the biosafety requirements. The QC plan should be designed to be compatible with the regulatory requirements.

## Professional Escalation Criteria

The risk-based QC plan should include the criteria for the professional escalation. The professional escalation is the process of the escalation of the issue to the supervisor or the manager.

The escalation criteria include:

- The failure of the control that is not resolved by the troubleshooting
- The failure of the assay that is not resolved by the troubleshooting
- The failure of the instrument that is not resolved by the troubleshooting
- The failure of the data analysis that is not resolved by the troubleshooting

The escalation criteria are used to ensure that the issues are resolved in a timely manner. The escalation criteria are used to ensure that the issues are resolved by the appropriate personnel.

## Building a Risk Register and Control Matrix for Molecular Assays

A risk-based QC plan becomes operational only when you translate identified risks into a structured, living document that guides daily decisions. The most practical tool for this translation is a risk register paired with a control matrix. The risk register captures every credible failure mode, its likelihood, its impact, and the controls assigned to detect it. The control matrix maps each control to the specific workflow step it monitors, the acceptance criteria that define a pass or fail, and the corrective action triggered by a failure. Together, these two documents convert the abstract principles of risk assessment into a working system that any operator can follow and any auditor can review.

### Structure of the Risk Register

The risk register is a table or spreadsheet with one row per identified failure mode. Each row contains the following fields:

- Failure mode identifier, such as FM-001, for unambiguous reference in corrective action reports
- Workflow step where the failure occurs, such as extraction, reverse transcription, amplification, library preparation, or variant calling
- Description of the failure, written in concrete terms that an operator can recognize
- Likelihood rating on a low, medium, or high scale based on observed data or validation records
- Impact rating on a low, medium, or high scale based on the effect on the final result
- Risk score calculated as the product of likelihood and impact, or as a matrix cell if you use a categorical scale
- Assigned controls that detect or mitigate the failure
- Responsible personnel for monitoring the control
- Review date and trigger conditions for reassessment

The likelihood rating should come from actual records where available. If your negative control log shows contamination in 2 of 120 runs over the past year, the likelihood is low. If the log shows contamination in 15 of 30 runs, the likelihood is high and the control strategy is clearly inadequate. The MIQE guidelines emphasize that assay performance parameters such as efficiency, limit of detection, and precision must be documented to establish a baseline for interpretation. The same principle applies to the risk register. Without baseline data, you cannot distinguish a rare event from a systemic problem.

The impact rating requires judgment about the consequence of a failure. A false-negative result in a diagnostic assay for an infectious agent has a high impact because it can lead to a missed diagnosis. A false-positive result in a research screen for a gene variant may have a lower impact if the result is confirmed by a second method. The impact rating should be assigned by the laboratory supervisor or the assay developer, not by the operator alone, because it requires an understanding of how the result will be used.

### Building the Control Matrix

The control matrix is the companion document to the risk register. It lists each control that the laboratory runs and specifies the following details. The control name, such as extraction negative control or sequencing positive control. The workflow step where the control is inserted. The type of control, such as positive, negative, internal, replicate, or calibration. The frequency of the control, such as every run, every batch, or every new reagent lot. The acceptance criteria that define a passing result. The corrective action to take when the control fails. The person responsible for reviewing the control result.

The acceptance criteria must be defined before the control is run, not after the result is available. For a positive control in a quantitative PCR assay, the acceptance criterion might be a threshold cycle value within a defined range based on the mean and standard deviation of the previous 20 runs. For a negative control, the acceptance criterion is the absence of amplification or the absence of sequence reads above a defined threshold. For an internal control, the acceptance criterion is a threshold cycle value or a read count that falls within the expected range for the sample type.

The corrective action should be specific and actionable. A generic instruction to investigate the failure is not sufficient. The corrective action should name the first steps to take, such as repeating the run with fresh reagents, checking the instrument calibration log, or reviewing the operator notes. The corrective action should also state when the failure must be escalated to a supervisor or to the assay developer.

### Implementing the Risk Register in Practice

The risk register should be created during the assay validation phase and updated after each significant change. The initial version is based on the failure modes that you identify during the workflow mapping exercise. The validation data provide the first set of likelihood ratings. The register is then reviewed after the first 20 to 30 runs to confirm that the likelihood ratings match the observed control failure rates.

The register should be reviewed at a defined interval, such as every six months, and after any of the following events. A new reagent lot that produces a control failure. A new instrument is installed or an existing instrument is serviced. A new operator is trained and begins running the assay. A new sample type is introduced. A control failure that is not resolved by the standard corrective action. A change in the intended use of the assay, such as moving from research to clinical use.

The review should compare the predicted likelihood ratings with the observed failure rates. If the observed rate is higher than the predicted rate, the control strategy is insufficient and the register should be updated. If the observed rate is lower than the predicted rate, the control may be over-specified and the frequency can be reduced, but only after the data support the change.

### Recording Control Results

The control results must be recorded in a format that supports both immediate review and long-term analysis. The minimum record for each control run includes the date, the operator, the instrument, the reagent lot numbers, the control result, and the pass or fail decision. The record should also include the corrective action taken if the control failed and the outcome of that action.

The records should be reviewed after each run to confirm that all controls passed. The records should also be transferred to a spreadsheet or a database for the statistical analysis. The Levey-Jennings chart and the Westgard rules require a continuous series of control results. The records must be complete and accurate for the statistical analysis to be meaningful.

The National Institute of Standards and Technology laboratory programs provide the measurement science that supports the calibration and the traceability of the instruments and the standards used in the assay. The NIST standards are used to calibrate the instruments and to validate the assays. The records should include the calibration dates and the results of the calibration to support the traceability of the measurements.

### Common Failure Patterns in the Register

The risk register will reveal patterns that are common across PCR and NGS assays. The most common pattern is contamination, which is detected by the negative controls. The contamination can be caused by the carryover of the amplicons, the contamination of the reagents, or the contamination of the environment. The register should include a specific failure mode for each source of contamination, because the corrective action differs. Amplicon carryover is addressed by the separation of the pre-amplification and the post-amplification areas. Reagent contamination is addressed by the replacement of the reagent lot and the review of the reagent handling. Environmental contamination is addressed by the cleaning of the workspace and the review of the operator technique.

The second common pattern is amplification failure, which is detected by the positive controls and the internal controls. The amplification failure can be caused by the inhibitors in the sample, the poor primer design, or the incorrect reaction conditions. The register should include a failure mode for each cause, because the corrective action differs. The inhibitors are addressed by the dilution of the sample or the use of a different extraction method. The poor primer design is addressed by the redesign of the primers. The incorrect reaction conditions are addressed by the optimization of the reaction.

The third common pattern is the base-call errors in the NGS data, which are detected by the sequencing run controls and the bioinformatics controls. The base-call errors can be caused by the sequencing instrument, the sequencing chemistry, or the data analysis. The register should include a failure mode for each cause, because the corrective action differs. The instrument errors are addressed by the calibration of the instrument. The chemistry errors are addressed by the use of the fresh reagents. The data analysis errors are addressed by the review of the bioinformatics pipeline.

### The Control Matrix as a Training Tool

The control matrix is also a training tool for the new operators. The matrix explains why each control is run and what the control is designed to detect. The new operator can use the matrix to understand the rationale for the QC plan, instead of simply following a list of steps. The matrix should be included in the training materials and the operator should be able to explain the purpose of each control before they are allowed to run the assay independently.

The training should also include the use of the risk register. The operator should be able to identify the failure mode that a control is designed to detect and the corrective action that is required when the control fails. The operator should be able to use the register to troubleshoot a control failure, instead of calling the supervisor for every issue.

### Escalation Criteria in the Register

The risk register should include the escalation criteria for the control failures that cannot be resolved by the standard corrective action. The escalation criteria are the following. A control failure that repeats after the corrective action has been applied. A control failure that is accompanied by the failure of the other controls in the same run. A control failure that is not explained by the operator notes or the instrument records. A control failure that occurs on a new reagent lot and is not resolved by the replacement of the reagents.

The escalation should be to the laboratory supervisor or the assay developer. The escalation should include the control records, the corrective action that was applied, and the results of the corrective action. The escalation should be documented in the risk register and the outcome should be recorded.

The risk register and the control matrix are the working documents that make the risk-based QC plan operational. They are the records that the laboratory uses to monitor the assay, to troubleshoot the failures, and to demonstrate that the QC plan is being followed. The documents should be reviewed and updated at the defined intervals and after the significant changes to the assay. The documents should be maintained as part of the laboratory records and should be available for the review by the supervisor, the auditor, or the regulator.

## Frequently Asked Questions

### What is the difference between a positive control and an internal control?

A positive control is a known template that is expected to produce a known result. The positive control is used to monitor the efficiency of the amplification. An internal control is a template that is added to every sample. The internal control is used to monitor the efficiency of the extraction and the amplification. The internal control is amplified with a different set of primers than the target.

### How often should the controls be run?

The controls should be run in every assay. The positive control and the negative control should be run in every assay. The internal control should be run in every sample. The frequency of the controls should be based on the risk of the assay. The higher the risk, the more frequent the controls.

### What is the Levey-Jennings chart?

The Levey-Jennings chart is a plot of the control results over time. The chart is used to detect the trends and the shifts in the assay performance. The chart is a plot of the control result on the time-axis and the time on the x-axis. The chart is used to detect the patterns that are not visible in the individual control results.

### What are the Westgard rules?

The Westgard rules are a set of rules for interpreting the control results. The rules are used to detect if the assay is in control or out of control. The rules are based on the mean and the standard deviation of the control results. The rules are used to detect the errors that are not visible in the individual control results.

### What is the role of NIST in the QC?

The NIST provides the measurement science that is the basis for the quality of the standards. The NIST provides the standard reference and the standards that are used to ensure the accuracy of the measurements. The NIST standards are used to calibrate the instruments and to validate the assays.

### What is the MIQE guidelines?

The MIQE guidelines are a set of guidelines for the reporting of the qPCR assays. The guidelines are published in Clinical Chemistry. The guidelines provide the list of the parameters that should be reported for the qPCR assays. The guidelines are used to ensure the transparency and the reproducibility of the qPCR assays.

### What is the risk of the contamination in the PCR?

The contamination is the most common failure pattern in the PCR. The contamination can be caused by the carryover of the amplicons, the contamination of the reagents, or the contamination of the environment. The contamination is detected by the negative controls. The contamination is prevented by the use of the separate areas for the extraction and the amplification.

### What is the role of the biosafety in the QC?

The biosafety is the set of the principles and the practices that are used to prevent the exposure to the biological agents. The biosafety is described in the Biosafety in Microbiological and Biomedical Laboratories. The biosafety is used to ensure the safety of the laboratory personnel and the environment. The biosafety is integrated with the QC plan to ensure the safety of the laboratory.

## Using the Evidence

| Source | Best use in this topic | Important limitation |
|---|---|---|
| [Molecular Biology and Laboratory Methods](https://www.ncbi.nlm.nih.gov/books) | official guidance | Check the linked page for current local requirements |
| [Biosafety in Microbiological and Biomedical Laboratories](https://www.cdc.gov/labs/bmbl/index.html) | official guidance | Check the linked page for current local requirements |
| [NIH Guidelines for Recombinant or Synthetic Nucleic Acid Molecules](https://osp.od.nih.gov/policies/biosafety-and-biosecurity-policy/nih-guidelines-for-research-involving-recombinant-or-synthetic-nucleic-acid-molecules) | official guidance | Check the linked page for current local requirements |

## Related Diagnostic Guides

- [Cell Migration Assays: Transwell vs. Wound Healing](/knowledge/diagnostics/emerging-tech/cell-migration-assays-transwell-vs-wound-healing)
- [Positive and Negative Controls in Diagnostic Assays: Selection and Interpretation](/knowledge/diagnostics/emerging-tech/positive-and-negative-controls-in-diagnostic-assays-selection-and-interpretation)
- [Biosafety Levels 1-4: A Comparative Framework for Diagnostic Laboratory Design](/knowledge/diagnostics/emerging-tech/biosafety-levels-1-4-a-comparative-framework-for-diagnostic-laboratory-design)
- [Cell Viability Assays: Choosing the Right Method for Your Experiment](/knowledge/diagnostics/emerging-tech/cell-viability-assays-choosing-the-right-method-for-your-experiment)
- [Choosing the Right NGS Library Preparation Kit: A Comparative Framework](/knowledge/diagnostics/emerging-tech/choosing-the-right-ngs-library-preparation-kit-a-comparative-framework)

## References and Further Reading

- [Molecular Biology and Laboratory Methods](https://www.ncbi.nlm.nih.gov/books). National Library of Medicine.
- [Biosafety in Microbiological and Biomedical Laboratories](https://www.cdc.gov/labs/bmbl/index.html). CDC and NIH.
- [NIH Guidelines for Recombinant or Synthetic Nucleic Acid Molecules](https://osp.od.nih.gov/policies/biosafety-and-biosecurity-policy/nih-guidelines-for-research-involving-recombinant-or-synthetic-nucleic-acid-molecules). National Institutes of Health.
- [Addgene Protocols](https://www.addgene.org/protocols). Addgene.
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> This article is educational and does not replace validated laboratory procedures, institutional biosafety review, manufacturer instructions, or professional interpretation.