A recent webinar presented by Dr. Jung Doh, senior applications scientist at Beckman Coulter Life Sciences, focuses on how the tissue fixation process affects RNA quality and influences downstream experiments such as next-generation sequencing (NGS). Providing solutions to overcome some of the challenges in RNA extraction from FPPE extractions, the webinar also answers your questions in the Q&A session.
This webinar covers:
• The differences between high, low and medium quality RNA
• The types of RNA quality metrics and methods of analysis used today
• Why it is difficult to isolate good-quality RNA from FFPE tissues
• The importance and effects of RNA quality and extraction methods in RNAseq workflows and outcomes
• Countermeasures to optimize FFPE sample preparations to enable successful downstream assays
Here are the highlights from the Q&A discussion at the end of the webinar:
Q: Can the extraction method influence the DV200 or is the quality of the RNA only dictated by the FFPE sample preparation?
JD: The sample preparation can have an effect on RNA integrity and quality, but also the extraction methodology used. Depending on how gentle and robust the kits are, you can get differences in RNA integrities with different RNA extraction methodologies. This is shown in some of the data we have shared. Watch the webinar on-demand >> It is important to note that this integrity has a direct impact on the sensitivity and specificity of the NGS result.
Q: Can you please elaborate on what the differences between the two extraction chemistries (A and B) are?
JD: In developing new extraction chemistries to improve NGS endpoints, we asked the question, ‘How does DV200 impact the NGS outcome?’ The data detailed in my webinar was produced as part of the development of our new FFPE extraction chemistry ‘FormaPure Total’ and FormaPure RNA. Kit A is a filter column-based kit, which applies vacuum or centrifuge to the samples being processed. Kit B, FormaPure RNA, uses a magnetic bead-based binding chemistry, so you add your buffers and sample to the beads, bind the nucleic acids and wash for impurities, then elute off the beads.
Q: With regards to the two extraction methods, which extraction chemistry was responsible for producing the higher quality RNA?
JD: FormaPure RNA
Q: How did you generate the RNA with different RNA integrity numbers (RINs)?
JD: The samples showing RNA with different integrity numbers were not generated; they are representative samples from the development work done in our R&D group. These RIN numbers were calculated directly from FFPE RNA extractions.
Q: Our fragment scans appear to have DNA contamination. I’ve never seen FFPE-derived RNA look like this on a fragment trace. Can you comment?
JD: I think this is pertaining to the traces that have higher molecular peaks. That is an excellent question because when we first saw the data, we thought it may be DNA carry-over or contamination. We tested this out experimentally and we found out it wasn’t actually DNA contamination. DNA contamination was found to be less than 0.01%. These peaks are degraded ribosomal RNAs which are typically not seen in FFPE RNA extracts. This speaks to the importance of using a robust and high performing extraction chemistry as well as the impact this chemistry can have on the NGS endpoints.
Q: Can we use DEPC treated water to resuspend RNA pellets?
JD: Yes, absolutely. DEPC treated water should not have an effect on the solution of RNA pellets.
Q: Why does the higher DV200 provide better sensitivity in NGS results?
JD: When you have a lower DV200 value, it means that a lot of your RNA fragments are below 200 nucleotides. Basically, those fragments that are below 200 nucleotides cannot be processed in your NGS library construction. When you have a lower DV200, you are losing a lot of the genetic information contained in the sample and potentially important information about the variant profile of the tissue.
Q: 1 µg is a lot of material for library construction. Why did you show data comparing 1µg when no one would do that?
JD: It’s not practical, most of the time, to get 1µg of RNA from FFPE samples. In this case, we asked the question, 'Can the NGS impact of poor-quality FFPE RNA be mitigated by loading more RNA into the library construction step?' Our dataset provides a pretty clear answer, showing more fusion calls with 5 times less input. Higher yield is clearly not a substitute for integrity.
Q: When extracting RNA, have you ever used TRIzol? How do you compare TRIzol with the kit you use to extract RNA?
JD: We did not use a TRIzol-based extraction method for comparison in this study. The core question we wanted to understand was not directly related to extraction but to RNA integrity. We would expect that if TRIzol extraction results in improved DV200, we would see the same positive impact on NGS endpoints. It is important to note, however, that TRIzol is associated with some very negative impacts to the overall workflow. Most importantly TRIzol is considered to be a very hazardous reagent to use and most labs try to mitigate hazardous materials when possible. In addition, TRIzol is extremely difficult to scale and not at all amenable to robust and reliable automation. Beckman focuses equal attention on performance, flexibility, scalability, and ease of use when developing new extraction solutions.