Direct integration with NGS library preparation methods
With truXTRAC, Covaris has developed a solution utilizing Adaptive Focused Acoustics (AFA) technology for high-efficiency extraction of nucleic acids from FFPE tissues. The robust and easy-to-use workflow ensures high-yields and enables direct integration with NGS and other molecular applications.
NGS-grade RNA and DNA from FFPE tissues
Active paraffin removal with truXTRAC, driven by AFA fluid microstreaming, promotes complete sample rehydration, thus improving tissue digestion by proteinase K and enhancing the dissociation of biomolecules for improved extraction of DNA or RNA.
IGV viewer sequencing coverage analysis for chromosome 19. FFPE kidney DNA extracted with Kit Q (red), Covaris truXTRAC (green) and for comparison fresh frozen kidney from the same source (blue). Coverage of >10x are indicated in dark colors, coverage of <10X are indicated in light colors.
Standardized and hands-free paraffin removal
With truXTRAC, paraffin is actively removed from FFPE tissue samples by the finely-controlled and reproducible acoustic energy delivered by AFA Focused-ultrasonicators. The entire process is performed in a single tube without transfer steps and last 5 minutes. DNA and RNA are routinely available for library preparation or other analytical methods in 3 hours.
Reproducible & operator independent process
Aqueous buffer, no organic solvent
Paraffin removal and tissue rehydration
Direct integration with NGS library preparation methods
Next Generation Sequencing applications require precisely controlled DNA fragmentation as part of the library preparation. Covaris AFA is the method of choice for DNA fragmentation in Genome Centers worldwide, because the DNA fragment size is easily controlled and highly reproducible. The DNA shearing step can be integrated into the truXTRAC workflow, greatly simplifying the library preparation protocol from FFPE tissue samples.
Centrifuge and Heat block microTUBE Adapters (quantity 10, PN 500406): Heat Block microTUBE Adapters are designed to fit a microTUBE into a heating block designed for 1.5 or 2 mL microcentrifuge tubes. It is required for Proteinase K incubation and to reverse formaldehyde crosslinks.
A short sequence showing paraffin emulsification from a 7 μm FFPE section
Improved FFPE DNA extraction for next generation sequencing using adaptive focused acoustics technology Ling Lin, et al ; 2014. “We found that Covaris had superior performance overall: (1) DNA yields were 6 times higher, and less specimens failed extraction: this is critically important when dealing with small tumor biopsies. (2) DNA was more readily available for PCR amplification. (3) Mean coverage of DNA fragments with high GC content was higher and more reproducible. (4) Library complexity was higher. In addition, the Covaris protocol required less hands-on time in the lab, and eliminated the need for hazardous organic solvents for paraffin removal.”
Genomic DNA Extraction Methods Using FFPE Tissue K Potluri, A Mahas, MN Kent, S Naik, M Markey – Analytical Biochemistry, 2015 “Of the three extraction methods, AFA samples showed increased amplicon length and decreased PCR failure rate. These findings support AFA as an improvement over previous DNA extraction methods for FFPE tissues.”
DNA Extraction of Lung Cancer Samples for Advanced Diagnostic Testing
Jeffrey Conroy et al, 2015 “FFPE tumor samples prepared using the Covaris truXTRAC isolation kit provides an efficient system for generating high quality DNA samples even from lung cancer specimens that previously failed testing with QIAGEN extraction. [..]. Additionally, DNA requirements for NGS based testing can be reduced 25-50% without loss of assay performance or analytic sensitivity as validated in our CLIA laboratory.”
Where can I find protocols for FFPE nucleic acid extraction?
A list of our current truXTRAC FFPE extraction protocols are published on our website. Click here and follow the link “FFPE Nucleic Acid Extraction” for a protocol that is related to your specific requirements.
Can I extract RNA or total nucleic acids using truXTRAC FFPE kits?
Yes, truXTRAC FFPE kits are available for RNA and total nucleic acids. Please look at our product page for further information.
Can I extract proteins using truXTRAC FFPE kits?
Currently extraction of proteins from FFPE tissue is under development. We are continually developing our protocols, so please check the product page on our website, under protocol tab, for the latest information.
Are the truXTRAC FFPE kits xylene-free?
Yes, Covaris Adaptive Focused Acoustics technology (AFA) allows for active paraffin removal in aqueous buffer, compared to the less efficient passive paraffin removal, which uses organic solvents.
What tissue sample types can be used?
FFPE sections mounted on slides, scrolls or cores are compatible with the truXTRAC FFPE protocols.
What is the maximum sample size for use with FFPE truXTRAC protocols?
The protocols are optimized for sections up to 25 µm in thickness and cores up to 1.2 mm in diameter.
What collection tools are recommended for sampling slide-mounted FFPE sections?
FFPE tissuePICK (PN: 520163) and FFPE tissuePICK Forceps (PN: 520164) should be used for FFPE sections ranging from 4 – 10µm in size. FFPE sectionPICK (PN: 520149) should be used for FFPE sections ranging from 7 – 10µm in size. The FFPE sectionWARMER (PN: 500403) is also available to facilitate collection using the FFPE sectionPICK. For more information on how to use these sample collection tools click here.
Why is my DNA/RNA yield low? How can I improve my yield?
Low yield might be caused by several different factors such as;
Low tissue to wax ratio in FFPE section: Excess paraffin will adversely affect the yield and quality of DNA and RNA extracted from FFPE tissue. We strongly advise trimming off any excess paraffin before sectioning a FFPE tissue block, or after the section has been cut from the FFPE block. A ratio of 80% tissue to 20% paraffin, or higher, is ideal. Repeat the procedure using additional sections until desired yield is achieved. In your initial use of the truXTRAC FFPE kit, use FFPE blocks that have been well characterized for yield and quality.
Insufficient tissue input: Increase FFPE tissue section thickness or use more sections up to 5mg total weight.
Proteinase K stored above recommended temperature or expired: Repeat the procedure using fresh Proteinase K. Always store Proteinase K solution at -20°C after resuspension.
If using bead purification low DNA yield may also be caused by;
Incorrect amount of beads were added: Ensure beads are resuspended well by vortexing beads thoroughly before each use.
Binding was incomplete: Make sure the correct reagent volumes were used and mix well before the 56°C incubation.
Beads lost during pipetting steps: Slowly draw up the supernatant into the pipet tip to not disturb the beads. It is normal for beads to stick to the pipet tips during the elution step, this has no effect on yield.
Why do I have no DNA/RNA?
Ensure that the protocol steps have been closely followed, including ethanol addition to wash buffers and mixing of the Proteinase K with the sample on the Covaris instrument. Proteinase K buffer contains glycerol, and may fall to the bottom of the microTUBE, therefore mixing is essential.
The concentration of DNA/RNA is too low, what could be the cause?
Elution volume may be too high. Repeat the procedure using lower elution volume (50 µl minimum volume is required for DNA and 20 µl for RNA). Alternatively, concentrate samples using ethanol precipitation or other means.
When following the FFPE DNA protocol with bead purification I notice clumping of magnetic beads during wash steps. Is this a problem?
Some clumping of magnetic beads may occur with specific FFPE blocks. Bead clumping will not affect the yield or purity of the DNA. If bead clumping is severe enough to clog pipette tips reduce the input sample amount.
Magnetic beads are sticking to the pipette tips during elution whilst following the FFPE DNA bead purification protocol. Will this affect my DNA yield?
Some sticking of magnetic beads to pipette tips may occur with specific FFPE blocks. Magnetic beads sticking to pipette tips during elution will not affect the yield or purity of the DNA. If bead sticking is severe enough to clog pipette tips reduce the input sample amount.
Why does my DNA not perform well in downstream applications such as qPCR?
DNA in FFPE sample blocks could be severely cross-linked or degraded, therefore ensure amplicons are designed to be as small as possible (<100 bp). DNA isolated using Covaris AFA technology is of the highest possible quality. Some FFPE sample blocks may be too degraded or cross-linked for some applications.
The DNA fragment size is too large when following Option A of the FFPE DNA protocol. What is the cause of this?
Too much emulsified paraffin in the sample absorbs some of the acoustic energy and will adversely affect DNA shearing efficiency. Trim any excess paraffin from tissue blocks before proceeding with the protocol. We recommend running a time course for DNA shearing (Step 7) to increase the treatment time by 30 second steps.
Which Covaris ultrasonicators are compatible with truXTRAC FFPE protocols?
All Covaris AFA instruments are compatible with truXTRAC FFPE kits. However each instrument will require specific accessories that may need to be purchased separately. Please look at the product page for detailed information.