3 Best Tips for Processing Plasma Cell-Free DNA & RNA

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3 Norgen scientists observing plasma/serum sample

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You are finally ready to start a new experiment with plasma or serum – congratulations! But what kind of data should you be expecting? Your results might surprise you, especially if you work with cellular sample types such as tissue biopsies.

This blog will cover our top 3 tips for working with plasma and serum for downstream cell-free nucleic acid analysis.

Tip #1: Handle with Care

It's not always possible to process blood samples immediately after collection, particularly when donors are at an ancillary site. During storage and transport of blood samples, hemolysis and white blood cell lysis are among the greatest concerns in terms of maintaining sample quality for cell-free nucleic acid analysis. When red and white blood cells begin to burst, either due to agitation during shipping or simply sitting on the bench for too long, their content will be released into the plasma or serum and can impair downstream cf-DNA and cf-RNA analysis.

  • Lysis of white blood cells will lead to cellular DNA and RNA released into the plasma, which can mask the low concentration of cell-free DNA and cell-free RNA and make sequencing more difficult and costly.
  • On the other hand, red blood cell lysis will release components such as hemoglobin into the plasma, which can co-elute with nucleic acids and inhibit PCR reactions and NGS performance.

Specialized preservation tubes can be a great solution to avoid hemolysis during transport and prevent the need for cold-chain storage. However, be sure you research any stabilizing tube well, as tubes containing fixatives (i.e. formaldehyde) can lead to cross-linking of DNA and other undesirable effects.

Hemolyzed plasma
Figure 1. No plasma hemolysis -----> Hemolyzed plasma (1)

Tip #2: Know what to Expect

If you are used to working with cellular samples, i.e. tissue biopsies, be prepared for your cell-free samples to look a little different upon analysis. Unprotected nucleic acids are quickly degraded in the blood by DNases and RNases. The surviving fragments of nucleic acids that persist in the circulation are protected in some form – usually as residual fragments bound to proteins (i.e. DNA wrapped around a nucleosome) or contained within protective packages (i.e. within extracellular vesicles).

Consequently, most cell-free DNA and cell-free RNA in plasma or serum will be under 200nt in length.

If you are finding high molecular weight fragments of DNA in your sample, it may be a sign that your plasma or serum was contaminated by cellular lysis during preparation. Similarly, significant 18S/28S ribosomal RNA peaks in a cell-free sample such as plasma/serum indicate that cellular contamination occurred.

Ensure the extraction kit you are using is designed to capture even the smallest DNA and RNA fragments. For example, many silica columns and magnetic bead systems have a molecular weight cutoff and will not efficiently capture the vast diversity of fragmented nucleic acids.

DNA Assay trace of cf-DNA isolated from 4ml Plasma
Figure 2. Agilent Bioanalyzer High Sensitivity DNA Assay trace of cf-DNA isolated from 4ml Plasma
Pico Assay trace of cf-RNA isolated from 200uL of plasma
Figure 3. Agilent Bioanalyzer RNA 6000 Pico Assay trace of cf-RNA isolated from 200uL of plasma

Tip #3: Check your Measurements

Since plasma and serum do not contain cells (if correctly prepared!), the amount of circulating nucleic acids present will be a fraction of what you would find in a tissue-based sample. Both species' concentration is typically very low and highly variable depending on the donor's health status. For example, the yield of cell-free DNA obtained from plasma in healthy individuals typically ranges from less than 1ng/mL to 100ng/mL. When comparing separate individuals, your yield will vary from person to person. You will also see a difference in yield for one individual depending on when you obtain your sample during the day. If you're looking to examine one specific subtype of cell-free nucleic acids downstream, i.e. circulating tumor DNA, it can feel like searching for a needle in a haystack!

Considering your elution will likely contain nucleic acids in the picogram/μL range, make sure you take the limit of detection of your quantification tools into account. Traditional spectrophotometer readings, which typically have an LoD of 10ng/μL, will not be able to provide an accurate measurement of yield or quality. Recent studies comparing quantification methods illustrate the significant variation in estimated yields between these tools, with spectrophotometry providing a significant overestimation of yield2. Consequently, qPCR is often suggested as the standard gold method for quantification.

How to use the right kit for processing Plasma Cell-Free DNA & RNA

In the tutorial below, we go over the step-by-step workflow for Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Mini Kit (cat. #55100).

If you’d like to discuss this topic or to know more about the kit mentioned above, please feel free to reach out to us at info@norgenbiotek.com

View References

References

  1. Jeon, Kibum, et al. "Quantification of cell-free DNA: a comparative study of three different methods." Journal of Laboratory Medicine and Quality Assurance 41.4 (2019): 214-219. LINK: https://www.jlmqa.org/journal/view.html?uid=1143&&vmd=Full

  2. Vioximiko, CC BY-SA 4.0 , via Wikimedia Commons


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