How do you troubleshoot and overcome background noise and contamination issues in your sample preparation?

1 Sources of noise and contamination

Noise and contamination can come from a variety of sources, such as the sample itself, reagents and equipment, the environment, and handling and storage of the samples. These sources can include non-specific binding of antibodies or probes to irrelevant targets, cross-reactivity or interference of reagents with each other or the sample matrix, carry-over or cross-contamination of samples or reagents during pipetting, centrifugation, or washing steps, contamination of reagents or equipment with microbes, dust, or other impurities, degradation or oxidation of samples or reagents due to improper storage or exposure to light, heat, or air, and human errors or variations in sample preparation protocols or techniques.

2 Detection of noise and contamination

Various methods can be used to detect noise and contamination, depending on the type and extent of the issue. For example, negative controls such as blank samples, no-template controls, or isotype controls can be used to assess the baseline noise or contamination level. Additionally, positive controls like known samples, standards, or spikes can be used to assess the recovery, sensitivity, and specificity of the sample preparation method. Comparing the results of different samples, replicates, batches, or methods can help identify any outliers, inconsistencies, or trends. Quality control checks like measuring purity, concentration, integrity, or activity of samples or reagents should also be performed. Finally, visualizing the samples or products with microscopy, electrophoresis, chromatography, or spectrometry can detect any anomalies, impurities, or artifacts.

3 Prevention of noise and contamination

Noise and contamination can be prevented by following good laboratory practices and sample preparation protocols. To achieve high specificity and efficiency, use high-quality, validated, and compatible reagents and equipment for your sample preparation method. Additionally, employ appropriate enrichment and depletion methods for your target molecules or cells. Utilize sterile, disposable pipettes, tubes, tips, and other accessories to avoid cross-contamination. Opt for filtered, deionized, or distilled water and solvents to prevent contamination with ions, metals, or microbes. When using aliquots of reagents and samples, store them at the recommended temperature and conditions to avoid degradation or oxidation. Wear gloves, lab coats, and other protective gear while working in a clean designated area to avoid contamination from the environment or yourself. Finally, follow the instructions and guidelines of the sample preparation protocol and document and standardize your steps and results.

4 Troubleshooting of noise and contamination

In order to troubleshoot noise and contamination, it is necessary to identify the source and cause of the issue, and then apply corrective or compensatory measures. Some potential solutions include changing or replacing reagents or equipment that are causing non-specific binding, cross-reactivity, interference, or contamination; increasing or decreasing the concentration, volume, incubation time, or temperature of the reagents or samples; adding or removing blocking agents, buffers, detergents, or stabilizers; repeating or modifying pipetting, centrifugation, or washing steps; diluting or concentrating samples or products; and using calibration curves, normalization factors, or statistical methods to correct for any bias or variation in the results.

5 Resources and references

Noise and contamination are unavoidable in sample preparation, however, they can be managed and reduced by utilizing the right methods and techniques. If you encounter any noise or contamination issues in your sample preparation, you can consult the manufacturer's website, manual, or technical support for your reagents and equipment. Additionally, you can look up the literature, databases, or forums for your sample type, target molecule or cell, and sample preparation method. Finally, you can reach out to experts, colleagues, or mentors in your field or laboratory for guidance.

6 Here’s what else to consider

This is a space to share examples, stories, or insights that don’t fit into any of the previous sections. What else would you like to add?