Nucleic Acid Quantification from FFPE Samples – Are You Doing it Right?
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The document discusses the challenges and methods of nucleic acid quantification from formalin-fixed, paraffin-embedded (FFPE) samples, highlighting the difficulties posed by factors such as fixation time and tissue type. It emphasizes the importance of selecting appropriate quantification technologies and automating the sample preparation process to ensure accurate results. The QIAxpert system and its spectral content profiling are presented as reliable solutions for quantifying nucleic acids in FFPE samples.
Nucleic Acid Quantification from FFPE Samples – Are You Doing it Right?
- 1. Sample to Insight Nucleic acid quantification from FFPE samples; are you doing it right? Marion Egli, Global Product Manager, QIAGEN Instruments AG Nucleic acid quantification from FFPE samples – September 2016 1
- 2. Sample to Insight Legal Disclaimer 2 QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease. For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor.
- 3. Sample to Insight Outline 3 The challenges of purifying DNA from FFPE samples1 Variations in DNA quantification results2 Impacts on downstream enzymatic reactions3 Approach for better sample insight4 Nucleic acid quantification from FFPE samples – September 2016
- 4. Sample to Insight Facts about FFPE 4 Current situation FFPE is a standard method for long-term preservation of tissue biopsies Large number of unprocessed FFPE samples are archived in tissue banks and biorepositories Samples are highly valuable, especially when they are well-characterized Needs Maximum DNA recovery from precious, small FFPE samples DNA must be suitable for all types of applications, including NGS Removal of co-purified RNA (i.e., for DNA sequencing) Sometimes there is no choice other than FFPE Nucleic acid quantification from FFPE samples – September 2016
- 5. Sample to Insight 5 But FFPE samples are tricky Nucleic acid quantification from FFPE samples – September 2016
- 6. Sample to Insight Typical challenges with FFPE samples 6 Effects of formalin fixation on DNA and RNA Nucleic acid quantification from FFPE samples – September 2016
- 7. Sample to Insight Prolonged fixation severely impacts your sample 7Nucleic acid quantification from FFPE samples – September 2016
- 8. Sample to Insight Different factors affect the nucleic acid quality 8 Sample handling Sample thickness Fixation time Incomplete dehydration Paraffin temperature Sample storage time & temperature Purification conditions Degradation of nucleic acid How to overcome these challenges? Learn more at QIAGEN.com/FFPE Nucleic acid quantification from FFPE samples – September 2016
- 9. Sample to Insight QIAamp DNA FFPE Tissue Kit 9 Kit specifications Silica membrane-based Up to 8 sections, each with a thickness of up to 10 µm and a surface area of up to 250 mm2 Purification of genomic DNA & mitochondrial DNA Elution volume 20–100 µl QIAcube protocol available Paraffin removal and sample lysis No need for overnight incubation Paraffin is dissolved in xylene and removed Sample lysis under denaturing conditions with proteinase K (1 h, 56°C) Incubation at 90°C to reverse formalin crosslinking Optional RNase treatment step Nucleic acid quantification from FFPE samples – September 2016
- 10. Sample to Insight Multiple studies investigating variation in FFPE sample processing 10 And a few open questions: Tissue type(s) not specified RNase digest – Yes/No unclear Relative yields vs. absolute yields Systematic deviations in quantification – dependent on method used “No method highly superior to others...” ... it is particularly important to choose the most reliable and constant DNA extraction system, especially when using small biopsies and low elution volumes... “ ...variation in pre-PCR steps is prevalent...” ...all common DNA quantification techniques can be used for downstream applications... “DNA quantitation may also impact PCR efficiency...” Conflicting messages in studies Nucleic acid quantification from FFPE samples – September 2016
- 11. Sample to Insight 11 What is the impact of this and what matters most? What factors contribute to variations in DNA quantity? Nucleic acid quantification from FFPE samples – September 2016
- 12. Sample to Insight Which factor has the highest impact on DNA quantity measured? 12 Study design Samples 5 different rat tissue types 2–3 different blocks 5–6x 3 sections, 10 µm thickness Assess variability caused by the samples themselves FFPE Samples Nanodrop QIAxpert Qubit In total 6000 data points! QIAcube Manual w/ RNase digest w/o RNase digest DNA purification QIAamp DNA FFPE Kit Automated using the QIAcube or manual processing With and without RNase digest Assess variability introduced by purification procedure (manual vs. automated) DNA quantification Using three different methods, 5 replicates/sample Assess variability caused by downstream quantification method Nucleic acid quantification from FFPE samples – September 2016
- 13. Sample to Insight DNA quantification technologies 13 Nanodrop QIAxpert Qubit Technology UV/VIS absorbance reading UV/VIS absorbance reading Fluorescence-based assay LOD 2 ng/µl (dsDNA) 1.5 ng/µl 10 pg/µl (assay-dependent) Sample volume 1 µl 2 µl 1–20 µl Samples per run 1 Up to 16 1 Drop-and-clean actions required Yes No No A260/280 Yes Yes A260/230 Yes Yes Nucleic acid quantification from FFPE samples – September 2016
- 14. Sample to Insight Comparison of different quantification systems 14 Concentration variability of purified FFPE samples What are the factors contributing to this variability when taking absorbance readings? QIAxpertNanodrop Qubit A huge variability with all UV/VIS-based systems Nanodrop shows the highest variance Qubit with the lowest variance Nucleic acid quantification from FFPE samples – September 2016 Nanodrop QIAxpert Qubit Sample no. 1500 3000 1500
- 15. Sample to Insight Does RNase treatment have an influence? 15 What about automated vs. manual processing? QIAxpertNanodrop Qubit Influence on the variance is related to RNA QIAxpert and Qubit show similar low variance on RNase-treated samples Nanodrop shows high variance on RNase- treated and untreated samples Concentration variability of RNase-treated FFPE samples with RNase digest without RNase digest Nucleic acid quantification from FFPE samples – September 2016
- 16. Sample to Insight Does the type of purification have an influence? 16 Concentration variability with type of purification QIAxpertNanodrop Qubit Kind of purification Automated Manual Kind of purification Automated Manual Kind of purification Automated Manual Higher level of standardization applying automated sample purification with RNase digest without RNase digest What role does the tissue type play? Nucleic acid quantification from FFPE samples – September 2016
- 17. Sample to Insight Comparison of different FFPE tissue types 17 Concentration variability among different FFPE tissue samples What role does the FFPE block play? 1. Rat Colon 2. Rat Heart 3. Rat Kidney 4. Rat Liver 5. Rat Muscle Different FFPE tissue material leads to different yield of nucleic acid Liver tissue is most challenging because of higher fraction of RNA Nucleic acid quantification from FFPE samples – September 2016 QIAxpertNanodrop Qubit
- 18. Sample to Insight Contribution to variability in quantification by the block/section 18 Higher differences in yields due to the quantification method chosen rather than the block Nucleic acid quantification from FFPE samples – September 2016 QIAxpertNanodrop Qubit
- 19. Sample to Insight What has the greatest influence on quantification of nucleic acids purified from FFPE samples? 19 Contribution Quantification technology 0.2816 Purification method 0.2569 Tissue type 0.2288 RNase digest 0.1424 FFPE block 0.0542 FFPE section 0.0362 The type of quantification technology matters the most! Nucleic acid quantification from FFPE samples – September 2016
- 20. Sample to Insight 20 Why does accurate quantity and purity assessment matter? Nucleic acid quantification from FFPE samples – September 2016
- 21. Sample to Insight Impact of improper quality control on downstream results 21 Fluorescence readingAbsorbance reading A method is required that accurately quantifies nucleic acids and detects impurities! - Sensitivity + No assay required + Temperature insensitive + Detection of contaminants (partly) + / - Total nucleic acid - No discrimination between DNA and RNA + Sensitivity - Assay required - Temperature sensitive + / - Specific quantification of target - No detection of other NA - No detection of contaminants Overestimation of DNA concentration Residual RNA undetected Weak or no enzymatic reactions Severe underestimation of DNA concentration due to DNA denaturation Residual other NA or contaminants undetected Inhibition of enzymatic reactions, false negatives Nucleic acid quantification from FFPE samples – September 2016
- 22. Sample to Insight 22 How to tell what’s really in the sample? Nucleic acid quantification from FFPE samples – September 2016
- 23. Sample to Insight DNA quantification technologies 23 Nanodrop QIAxpert Qubit Technology UV/VIS absorbance reading UV/VIS absorbance reading Fluorescence-based assay LOD 2 ng/µl (dsDNA) 1.5 ng/µl 10 pg/µl (assay-dependent) Sample volume 1 µl 2 µl 1–20 µl Samples per run 1 Up to 16 1 Drop-and-clean actions required Yes No No A260/280 Yes Yes A260/230 Yes Yes Discriminate between molecules of interest No Yes (Yes) Nucleic acid quantification from FFPE samples – September 2016
- 24. Sample to Insight 1. Absorbance measurement (and background correction) 2. Content profiling of the measured spectrum by fitting of reference spectra into: • Specific DNA or RNA spectrum • Impurities spectrum • Residue spectrum 3. Quality control • Impurities spectrum • Residue spectrum • Background spectrum • A260/A280 ratio for protein contamination Spectral content profiling with QIAxpert 24 RNA SaltsDNAExamples of reference spectra ? unknown 1000 ng/µl total NA vs. 750 ng/µl DNA Nucleic acid quantification from FFPE samples – September 2016
- 25. Sample to Insight Remember this one? 25 What would it look like with spectral content profiling? QIAxpertNanodrop Qubit Concentration variability of RNase-treated FFPE samples with RNase digest without RNase digest Nucleic acid quantification from FFPE samples – September 2016
- 26. Sample to Insight Results with QIAxpert spectral content profiling 26 Concentration variability of RNase-treated FFPE samples QIAxpert A260Nanodrop QubitQIAxpert SCP with RNase digest without RNase digest Nucleic acid quantification from FFPE samples – September 2016 What is reported by QIAxpert?
- 27. Sample to Insight QIAxpert – telling DNA from RNA without a dye 27 Classic absorbance reading Spectral content profiling QIAxpert tells you what’s really in the sample Nucleic acid quantification from FFPE samples – September 2016
- 28. Sample to Insight QIAxpert maximizes the sample insight Nucleic acid quantification from FFPE samples – September 2016 28 Quantifying molecule of interest vs. other nucleic acids Value for DNA Value for total NA QIAxpert SCPNanodrop Qubit
- 29. Sample to Insight 29 Conclusions Nucleic acid quantification from FFPE samples – September 2016
- 30. Sample to Insight Summary 30 FFPE tissue samples present a number of challenges If you really want to be sure that the genomic DNA you quantify represents what is in your sample: Choose your quantification technology carefully Automate your sample prep (i.e., using a QIAcube) Apply a RNase digestion step Be aware of systematic differences between technologies when quantifying nucleic acids QIAxpert system & spectral content profiling offers reliable quantification of FFPE samples QIAGEN provides a number of solutions – from Sample to Insight – supporting your research efforts using FFPE samples Nucleic acid quantification from FFPE samples – September 2016
- 31. Sample to Insight 31 Thank you for your attention! Questions? Learn more about our Quality Control Solutions at www.qiagen.com/QCSolutions For up-to-date licensing information and product-specific disclaimers for QIAGEN products, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor. Nucleic acid quantification from FFPE samples – September 2016
Editor's Notes
- #12: http://biomarkerinsights.qiagen.com/2015/10/23/bb-dna-quantity-ffpe/