EZ2 AllPrep DNA/RNA FFPE Kit

For automated and simultaneous purification of DNA and RNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples

S_1151_7_EZ2_DNA_RNAAllPrepFFPEKit

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EZ2 AllPrep DNA/RNA FFPE Kit (48)

Cat. No. / ID:  954734

For 48 preps: EZ2 AllPrep DNA/RNA FFPE cartridge, disposable filter-tips and tip-holders, tubes, Paraffin Removal Solution, Proteinase K, DNase, RNase-free water and buffers
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₩1,060,000.00
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The EZ2 AllPrep DNA/RNA FFPE Kit (48) is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

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Features

  • Patented technology to extract DNA and RNA from the same sample – no need to divide the sample
  • Elution of DNA and RNA in separate fractions offers convenient downstream analysis
  • Reduces sample consumption
  • Optimizes crosslink removal for both DNA and RNA
  • No overnight proteinase K incubation

Product Details

The EZ2 AllPrep DNA/RNA FFPE Kit is used with the EZ2 Connect instrument. It automates the extraction of both DNA and RNA, from exactly the same piece of sample in two distinct elution fractions.

The kit technology allows the reversal of crosslinking caused by the sample’s exposure to formalin without overnight proteinase K incubation.

Performance

The EZ2 AllPrep DNA/RNA FFPE Kit is designed for automated purification of DNA and RNA from FFPE samples, using magnetic-bead technology and prefilled cartridges.

The automated workflow is presented in figure " Automated, simultaneous DNA and RNA purification from FFPE samples." This workflow provides a greater degree of automation (figure
" Degree of automation") and provides superior nucleic acid yields from FFPE samples compared to manual workflows and workflows from other suppliers (figure " DNA and RNA yields from spleen FFPE.", figure " DNA and RNA yields from human spleen.", figure " RNA and DNA yield from rat kidney and heart." and figure " Comparison of manual and automated extraction."). The decrosslinking step improves efficiency and library complexity (figure " Decrosslinking efficiency." and figure
" Library complexity.")

See figures

Principle

DNA and RNA extraction from the same sample is needed to compare genomic and transcriptomic data reliably. Usually, this is done by splitting the sample into two separate reactions/tubes/containers. The extractions are then processed separately. Thus, DNA is extracted from one piece of the sample, while RNA is extracted from the other piece.

Conventional methods have two major disadvantages: First, the amount of nucleic acids that can be extracted from the sample is cut into half. Second, because DNA is extracted from one group of cells, and RNA is extracted from a separate group, there can be differences in the properties of the cell sources.

The patented AllPrep DNA/RNA FFPE procedure incubates FFPE tissue samples in a lysis buffer that releases RNA into the supernatant and leaves DNA in the pellet. The RNA and DNA are then purified in separate tubes but within the same automated run using one cartridge in the EZ2 Connect instrument. This way, DNA and RNA can be isolated from as many as 24 samples, all at the same time.

Crosslink removal

Extended crosslink removal at high temperature reverses more crosslinks, but it also increases nucleic acid fragmentation. RNA is more susceptible to heat-induced fragmentation than DNA – but it is also less affected by crosslinks.

Therefore, the optimal conditions for decrosslinking DNA and RNA are different.

By generating separate lysates for RNA and DNA, the EZ2 AllPrep DNA/RNA FFPE workflow allows optimal crosslink removal conditions for each nucleic acid type.

Procedure

EZ2 AllPrep DNA/RNA FFPE technology solubilizes and incubates a single FFPE sample to release RNA into the supernatant while DNA is left in the pellet. The supernatant is incubated to remove RNA crosslinking. Finally, the tubes that separately contain the supernatant and pellet(s) are transferred to EZ2 Connect.

The EZ2 Connect automates all remaining steps of the workflow. These include an additional lysis and crosslink-removal step for DNA, binding of nucleic acids to magnetic particles, removal of contaminants with multiple washing steps, and elution of DNA and RNA into different tubes (see EZ2 AllPrep DNA/RNA FFPE workflow).

See figures

Applications

DNA and RNA purified with the EZ2 AllPrep DNA/RNA FFPE Kit can be used for real-time PCR, Pyrosequencing and other downstream applications.

Supporting data and figures

Specifications

FeaturesSpecifications
Applications(RT-)PCR, (RT-)qPCR, NGS, genotyping, microarray analysis
Elution volumeRNA: 50–60 µl; DNA: 100–120 µl
Purification of total RNA, miRNA, poly A+ mRNA, DNA or proteinRNA and DNA
TechnologyMagnetic particle technology
Number of preps per run1–24 samples per run
Main sample typeFFPE tissue samples
Sample amountmax. 4*10 µm sections or 2*20 µm sections
YieldVaries depending on sample type, as well as on storage and fixation conditions

Resources

Kit Handbooks (1)
Scientific Posters (1)
Eva Haenssler, Simon Hertlein, Christina Fischer, Nicole Pickave, Martin Schlumpberger
QIAGEN Strasse 1, 40724 Hilden, Germany
Technical Information and Important Notes (1)
Quick-Start Protocols (1)
Safety Data Sheets (1)
Certificates of Analysis (1)

FAQ

When should I use the ‘Standard’ vs. the ‘Fast’ protocol for the EZ2 AllPrep DNA/RNA Kit
The ‘Standard’ version of the EZ2 AllPrep DNA/RNA FFPE protocol provides optimized DNA decrosslinking and PCR performance. However, it can negatively impact DNA integrity and potentially NGS applications. In contrast, the ‘Fast’ version yields less fragmented DNA.
FAQ ID - 3948
What size of RNA can be expected?
This can vary significantly and strongly depends on the RNA quality present in the original FFPE sample. Formalin-fixation, paraffin-embedding and storage conditions are factors that affect the RNA size distribution and may cause significant fragmentation of nucleic acids. To limit the extent of nucleic acid fragmentation, be sure to:
Fix tissue samples in 4%–10% formalin as quickly as possible after surgical removal, use a fixation time of 14–24 h (longer fixation leads to more fragmentation, reducing performance in downstream assays).
Thoroughly dehydrate samples prior to embedding (residual formalin can inhibit proteinase K digestion)
Store FFPE tissue samples at -20°C, if possible.
FAQ ID - 3954
An immediate subsequent AllPrep DNA/RNA FFPE run does not start right away.
Make sure that the heating block is at room temperature before starting your run. This can be done by starting the ‘Cooling’ protocol from the EZ2 Connect main screen.
FAQ ID - 3952
Why is there a need to overlay the resuspended DNA pellet with PRS again (step 17 of QSP)?
The purpose of the PRS in this step is to prevent evaporation of the buffer ATL and Proteinase K that had been added to the DNA pellet during the heating procedures of the EZ2. Therefore, the PRS serves a dual function in this kit, not only deparaffinization.
FAQ ID - 3951
Can I use the EZ2 AllPrep DNA/RNA FFPE kit to extract DNA/RNA from fresh frozen tissue?
Unfortunately, the EZ2 AllPrep DNA/RNA FFPE Kit cannot be used for fresh-frozen tissue as its chemistry relies on formalin-fixed tissue. However, there is an option for isolation of total nucleic acids using the EZ1&2 RNA Tissue Mini Kit; https://www.qiagen.com/de/products/discovery-and-translational-research/dna-rna-purification/rna-purification/total-rna/ez1and2-rna-tissue-mini-kit/ on the EZ2. In this case, both RNA and DNA would end up in the same elution tube. The protocol is available on our webpage https://www.qiagen.com/de/resources/resourcedetail?id=657cc5b3-6e8e-4e48-b730-ff1204c3320b&lang=en. If you would need to have RNA and DNA in separate tubes, the only solution would be to use two different kits, the RNA and the DNA Tissue kit. The limitation here is of course that RNA and DNA would not be extracted out of the exact same piece of tissue but would require splitting up the sample and performing two separated runs on the instrument.
FAQ ID - 3953
The integrity of my DNA sample (e.g. QIAxcel Connect) is bad.
Make sure to use the ‘Fast’ version of the EZ2 AllPrep DNA/RNA FFPE protocol as this provides less fragmented DNA. However, this can negatively impact its performance in other downstream applications such as qPCR.
FAQ ID - 3958
Are there other options for paraffin removal?
Deparaffinization with the supplied Paraffin Removal Solution is recommended, however, other standard methods such as heptane and methanol or xylene can be used as well. After any deparaffinization method, proceed with step 7 of the Quick Start Protocol for the EZ2 AllPrep DNA/RNA Kit.
FAQ ID - 3949
My overall RNA yield is fine but RT-PCR performance poor – how can I improve this?
This might indicate a higher degree of crosslinks still existing in the purified RNA. Extending the RNA decrosslinking step (QSP step 12) to up to 60 min can improve RT-PCR performance. However, this may impact RNA integrity which is considered a crucial parameter for other downstreams like NGS.
FAQ ID - 3956
Are there specific recommendations for performing RT-PCR on RNA isolated from paraffin-embedded samples?
Paraffin-embedded or fixed samples typically yield fragmented, partially degraded RNA. In addition, RNA quality will depend greatly on the handling of the samples before, during, and after the fixation procedure. 
If performing RT-PCR with degraded RNA, we recommend for cDNA synthesis the use of gene-specific primers or random nonamers rather than oligo-dT primers, since the mRNA poly-A tail may have been lost due to degradation. Regarding PCR primer design, shorter amplicon sizes are preferable.
FAQ ID - 3955
What are recommended stopping points in the procedure of the EZ2 AllPrep DNA/RNA FFPE Kit?
After cutting the sections and transferring them into a safe-lock tube (end of step 3 in QSP), samples can be stored at -20°C for several days. Before proceeding, samples should be equilibrated to room temperature in this case.
After separation of RNA lysate and DNA pellet (end of step 11), both RNA lysate and DNA pellet can be stored at -20°C for several days. Before proceeding, samples should be thawed at room temperature.
FAQ ID - 3950
Which version of the EZ2 Connect can run the EZ2 AllPrep DNA/RNA FFPE Kit?
All versions of the EZ2 Connect (EZ2 Connect, EZ2 Connect Fx and EZ2 Connect MDx) can run the kit.
FAQ ID - 3947
Why are my DV200 values of my RNA sample so low?
Compared to some other methods, the EZ2 AllPrep DNA/RNA FFPE protocol also binds small RNA fragments which are abundantly present especially in fragmented samples from FFPE tissue. This can cause a low DV200 value but should not be considered as measure of bad quality of your RNA.
FAQ ID - 3957