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Isolation of DNA from FFPE samples without paraffin removal
Suzanne Kennedy, Ph.D., Director of R&D
Sponsored,vendor-submitted protocol    Sponsored by Mo Bio Laboratories, Inc.    Published in November 2009 (p.21) DOI: 10.2144/000113291

Abstract

Isolation of DNA from paraffin-embedded tissue remains an arduous task for researchers screening hundreds of samples for genetic analysis. Removal of the wax coating encasing the thin layer of tissue and extraction of sufficient intact DNA for use in PCR and qPCR are major obstacles to working with these precious samples. A new method that allows for high yields of DNA from formalin-fixed, paraffin-embedded (FFPE) samples without paraffin removal is described. Increased sensitivity in qPCR is the result.

Introduction

High yields and purity are the main problems resulting in poor performance using DNA extracted from FFPE tissues. To overcome these obstacles for successful PCR, a method was developed that avoids the use of xylene for paraffin removal and thus eliminates extra handling steps that can lead to sample loss. The BiOstic® approach digests tissues directly through wax, allowing for higher yields of intact DNA. The direct lysis method has been compared side by side with a competitor’s xylene deparaffinization method and was found to provide superior yields and purity based on Nanodrop and qPCR data.

Materials

BiOstic® FFPE Tissue DNA Isolation Kit (Cat. no. 12250-50; MO BIO Laboratories)

FFPE tissue slices (ILSbio, LLC, Chestertown, MD, USA)

Methods

Figure 1. qPCR was performed using DNA purified from the BiOstic® Kit and the Qiagen QIAamp DNA Mini Kit following their protocols for FFPE tissues. A 74-bp primer set with a hydrolysis probe for GAPDH (Applied Biosystems Inc, Foster City, CA, USA) was used with 100 and 10 ng of template. A standard curve was generated using DNA purified from human samples resulting in an amplification efficiency of 101% (slope -3.28). No amplification was observed in the non-template control.

1. Add Solution FP1 and FP2 to the tube containing up to 15 mg of FFPE tissue slices.

2. Add Solution FP3 and heat at 55ºC for 1 h followed by 90ºC for 1 h.

3. Centrifuge to separate the debris from the digested lysate and transfer to a clean tube.

4. Add Solution FP4 and Solution FP5 and mix. Load the mixture onto a spin filter and centrifuge to bind the DNA to the silica membrane.

5. Wash the spin filter with Solution FP6. Decant the flow-through and wash with Solution FP7.

6. Discard the flow-through and centrifuge for 2 min to dry the membrane. Transfer to the final elution tube.

7. Elute the purified DNA in 50-100 L Solution FP8. DNA is now ready for use in qPCR or PCR applications.

Results





Figure 2. FFPE tissue extraction of patient GIST samples using the BiOstic® or competitor’s kit. The red bars indicate that the overall yields of DNA from the MO BIO kit were consistently higher for all samples tested. Data was provided by an external lab. DNA was extracted from normal human kidney FFPE tissue (2 slices, each 10 m thick) following the BiOstic® FFPE Tissue DNA Isolation Kit standard protocol and with a competitor kit’s recommended protocol for FFPE tissues. The competitor’s method uses xylene to deparaffinize the tissue followed by ethanol washes to remove the xylene. These additional steps can result in residual chemical carryover and loss of tissue. Results demonstrate that the non–paraffin removal method increases the detection sensitivity of the GAPDH gene (Figure 1). A one-cycle increase in detection (30 for BiOstic® vs. 31 for the competitor) equates to a two-fold increase in sensitivity. Even small increases in sensitivity can enhance the reliability and accuracy of quantitative results, especially when only one slice is available for processing.

To analyze the efficiency of extraction from patient tumor samples, the BiOstic® FFPE Tissue DNA Isolation Kit was evaluated by an external diagnostic lab. Gastrointestinal stromal tumor (GIST) FFPE tissue samples were removed from slides post-histology and extracted using the xylene-free BiOstic® kit or a competitor’s method that requires the use of xylene. Single slices of 10-m thickness were processed per prep, and overall DNA yield was measured on the Nanodrop (NanoDrop products, Wilmington, DE, USA). The BiOstic® kit gave consistently higher yields of DNA over the competitor’s kit (Figure 2).

Summary

FFPE tissue samples have inherent difficulties that make extraction a challenge. Removal of any steps that reduce yields and purity is advantageous. The BiOstic® FFPE Tissue DNA Isolation Kit provides a way to obtain maximal DNA yields from as little as a single slice of tissue, and the resulting DNA is suitable for genotyping analysis. No other method provides purified DNA faster or easier.

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