Retrieval of usable nucleic acids from FFPE samples

Retrieval of usable nucleic acids from FFPE samples

The ideal starting material for nucleic acid purification from FFPE tissues are freshly-cut sections. Omit the first 1–2 sections because they are prone to oxidation of DNA caused by exposure of the block to the atmosphere. Due to this potential oxidation, freshly-cut sections should be processed immediately and not stored. If storage cannot be avoided, make sure to store the paraffin sections at lower temperatures, preferably at –20°C or –80°C. Additionally, use 5–10 μm sections for best results. Thicker sections may result in lower nucleic acid yields, even after prolonged incubation with proteinase K.

Figure 4. Key steps. Nucleic acid sample preparation from FFPE samples.
Figure 5. Efficient deparaffinization. Deparaffinization of 2x10 µm FFPE sections from human lung and human liver was carried out either by treatment with the QIAGEN Deparaffinization Solution or by melting of paraffin before lysis. Genomic DNA was purified using the QIAamp® DNA FFPE Tissue Kit. DNA yields were measured using UV-Vis-based (QIAxpert®) and fluorometric technologies (Qubit®). The same volume of the diluted eluate was used as template in a PCR assay amplifying a 66 bp amplicon of the 18S rDNA. Samples treated with Deparaffinization Solution show significantly higher yields and lower Cq values than samples where paraffin was melted. This indicates that inefficient deparaffinization leads to insufficient release and poor recovery of DNA. 
FFPE samples contain DNA molecules that are crosslinked mainly to protein molecules. Such cross-linked DNA is less efficiently recovered during the purification procedure and is a poor substrate for PCR and other enzymatic assays. Therefore, breaking these crosslinks is a necessary step during gDNA purification. Crosslink remnants like nucleic acids attached to proteinase K-digested protein fragments should also be reversed if possible, as they can lead to sequence artifacts during DNA analysis. However, decrosslinking and reversing chemical modifications should be carried out with care, because harsh reaction conditions may result in further DNA fragmentation. For efficient decrosslinking, incubate at elevated temperatures such as 90°C for 1 hour. Alternatively, incubate longer at slightly lower temperatures, for example, 4 hours at 80°C, for probably gentler yet equally efficient decrosslinking. However, overnight incubation at 56°C is not recommended because, without crosslink removal at higher temperatures, performance in downstream qPCR remains poor (Figure 6B). Also, UV-Vis-based quantification was unreliable for such samples (Figure 6A).
Figure 6. Efficient decrosslinking. 2 x 10 µm FFPE sections from human lung were treated with proteinase K at 56°C for 1h and then incubated as indicated. Genomic DNA was purified using the QIAamp DNA FFPE Tissue Kit. DNA yields were measured using UV-Vis-based (QIAxpert) and fluorometric technologies (Qubit). The same amount of DNA was used as template in a duplex PCR assay amplifying a short (66 bp) and a long amplicon (500 bp) of the 18S rDNA. A: Incubation for 1h at 90°C and 4h at 80°C achieved similarly low Cq values, in contrast to the shorter incubations at 80°C or incubations at lower temperatures. Hence, shorter incubations at high temperatures or longer incubations at slightly lower temperatures work best for efficient decrosslinking.
Crosslinking and chemical modifications also affect RNA. Since RNA is more easily fragmented by heat treatment, optimized buffer conditions and lower incubation temperatures are critical. While incubation above 80°C may slightly improve RNA performance in RT-PCR further, it will also result in greater RNA fragmentation. On the other hand, incubation at lower temperatures will result in lower yields and significantly poorer performance in RT-PCR and other applications. Isolation of short RNA in a separate fraction to enrich miRNA (and other short RNA) is usually not feasible from FFPE specimens, because of the presence of short fragments derived from long RNA species.
Purification of DNA and RNA from FFPE sections allow reliable comparison of genomic and transcriptomic data. This is particularly important when working with tumor tissues as they often contain a heterogeneous distribution of healthy and malignant cells, which means different sections from the same sample may differ in their cellular composition. Simply dividing a sample in half for separate DNA and RNA purification procedures results in the purification of DNA and RNA from different populations of cells, which may differ in their properties. Simultaneous purification of DNA and RNA from the same sample prevents wastage of precious FFPE samples.
Did you know? Recent advances in NGS chemistries, platforms and bioinformatics pipelines have empowered users to efficiently interrogate DNA and RNA modifications in biological samples. Current approaches, however, require the use of two separate library prep workflows - one each for DNA and RNA. QIAseq® Multimodal Panels overcome these limitations by consolidating targeted DNA and RNA enrichment and analyses. Unlike current approaches, QIAseq Multimodal Panels do not require two separate workflows for DNA and RNA analyses, saving time and limited samples.
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