Normalizing DNA and RNA – Throw in Automation for Equal Measure

In molecular biology assays, such as next-generation sequencing and PCR, normalization is an essential but tricky step to generate reliable and comparable data. Read on to find out more about normalizing DNA (or RNA) and how to improve speed and reliability by automation.

When analyzing DNA or RNA, normalization can sometimes seem insignificant compared to obtaining your precious samples and analyzing the data. Nevertheless, many assays would be impossible or highly impractical without a normalization step somewhere down the line.

In PCR for example, DNA concentration from biopsies or cell-based in vitro assays is routinely checked after extraction using spectrophotometry. These values help scientists ensure that DNA quantities are as consistent as possible when setting up a PCR plate, which improves precision and avoids wasting reagents.

In next-generation sequencing (NGS), normalization usually occurs at several stages of the protocol. Examples include supplying sufficient input material after DNA (or RNA) extraction or balancing libraries after library prep. Both normalization steps help to obtain consistent, reliable data when working with multiple samples by ensuring equal distribution of data. The former is usually based on straightforward spectrophotometry and the latter on qPCR.

Pipette, adjust, pipette, repeat…

In PCR, normalization involves assessing the concentration of each sample, calculating the appropriate volume of sample and buffer needed (usually on a spreadsheet), and pipetting these volumes for the next step of the protocol. When doing NGS, libraries are normally normalized as part of library pooling by ensuring equimolar amounts of each library in the pooled sample. The pipette needs to be adjusted multiple times for every pooled sample: first for each library, then for the buffer or diluent. This process is then repeated for every sample, potentially hundreds in a single experiment.

The result of these repetitive pipetting-and-adjusting steps is that normalization is often time-consuming and tedious. It’s also prone to pipetting errors as the researcher needs to check the required volume for every sample on the spreadsheet and adjust the dial on one or multiple pipettes.

Another challenge of manually adjusting the concentration for each sample is the strain on the hands and fingers of the user. Even with modern ergonomic pipettes, carrying out these steps, and opening and closing the tube lids, quickly becomes uncomfortable – and can even lead to injuries.

Don’t wait, automate

To take the strain out of normalization, molecular biology labs have the option of switching to automated liquid handling. These systems carry out precise pipetting – unattended and without breaks or interrupting phone calls. These systems often come with specialized tools for guiding the user through normalization, meaning that they can help labs reduce overall preparation time as well as hands-on time.

At the preparation stage, automated liquid handling systems can make it straightforward for users to obtain accurate pipetting volumes with a low risk of error. By using well known data formats, Excel and csv files for instance, it is easy to convert measurements into automated pipetting protocols. After completion, these systems can produce a log file confirming every pipetting operation for added reliability.

Less pipetting, faster results

When it comes to the pipetting itself, automated liquid handling systems benefit users in several ways. First, there’s the operator’s time; automation works fast and unattended, greatly reducing time spent on normalization. For example, normalizing an experiment consisting of 96 samples can be carried out with automated liquid handling, freeing up 50 minutes of time.

Switching to automated pipetting also has important ergonomic benefits for lab users. Because of the constant adjustments required for each sample, it’s one of the more demanding tasks for a user’s hands – and that’s in an already pipetting-heavy PCR or NGS protocol. Automation dramatically reduces the number of pipetting operations for the user during normalization.

Automated liquid handling systems also offer the benefit of an enclosed space. Cleanliness and consistency are key attributes of PCR and NGS protocols and working in a closed system reduces contamination risks that can affect the results. When working with equipment is fitted with cooling elements, researchers can keep their samples at a low temperature without needing an ice machine. These factors work together to take the burden out of normalization and make sure samples are pure, consistent, and ready for PCR, library prep, or sequencing.

Eppendorf offers a range of products that support and automate PCR and NGS workflows. Using its long history of making precise, accurate manual pipettes and transparent tips, Eppendorf has produced the epMotion® automatic liquid handling systems, optimized for PCR and NGS tasks – as well as the Mastercycler® X50 and Eppendorf twin.tec® LoBind PCR plates®. These advanced solutions help ensure accurate molecular biology results with low risk of contamination in a fast, efficient workflow.

To find out more about how Eppendorf supports scientists working in NGS, take a look here.