Nucleic acid extraction from formalin-fixed paraffin-embedded cancer cell line samples: a trade off between quantity and quality?
© The Author(s). 2016
Received: 12 February 2016
Accepted: 28 October 2016
Published: 14 November 2016
Advanced genomic techniques such as Next-Generation-Sequencing (NGS) and gene expression profiling, including NanoString, are vital for the development of personalised medicines, as they enable molecular disease classification. This has become increasingly important in the treatment of cancer, aiding patient selection. However, it requires efficient nucleic acid extraction often from formalin-fixed paraffin-embedded tissue (FFPE).
Here we provide a comparison of several commercially available manual and automated methods for DNA and/or RNA extraction from FFPE cancer cell line samples from Qiagen, life Technologies and Promega. Differing extraction geometric mean yields were evaluated across each of the kits tested, assessing dual DNA/RNA extraction vs. specialised single extraction, manual silica column based extraction techniques vs. automated magnetic bead based methods along with a comparison of subsequent nucleic acid purity methods, providing a full evaluation of nucleic acids isolated.
Out of the four RNA extraction kits evaluated the RNeasy FFPE kit, from Qiagen, gave superior geometric mean yields, whilst the Maxwell 16 automated method, from Promega, yielded the highest quality RNA by quantitative real time RT-PCR. Of the DNA extraction kits evaluated the PicoPure DNA kit, from Life Technologies, isolated 2–14× more DNA. A miniaturised qPCR assay was developed for DNA quantification and quality assessment.
Careful consideration of an extraction kit is necessary dependent on quality or quantity of material required. Here we provide a flow diagram on the factors to consider when choosing an extraction kit as well as how to accurately quantify and QC the extracted material.
KeywordsFFPE DNA RNA Manual Automated Extraction
Highly multiplexed assays, capable of profiling many genetic biomarkers in a single experiment, are of rising importance in the field of life sciences, enabling mapping of entire biological pathways [1–5]. Our rapidly growing understanding of the molecular mechanisms driving disease progression enables acceleration of personalised medicines into the clinic, particularly in oncology [6–8]. Such assays require sufficient quantities of high quality DNA and RNA extracted from clinically relevant patient samples.
Formalin is the most widely used fixative, used for over a century by hospitals to preserve clinical samples for long term storage. Extensive collections of formalin fixed paraffin embedded (FFPE) clinical samples exist worldwide, representing an invaluable resource for prospective and retrospective studies on archival tissue [9, 10]. However, multiple factors influence the efficiency of formalin fixation including tissue size, fixation temperature and duration, and the amount of time that elapses before the sample is fixed . The lack of standardised procedures for collecting and processing tissue samples results in a range of FFPE qualities across different sites which can be further influenced by the age of the tissue block . Scientists are constantly faced with the challenge of obtaining sufficient amounts of high quality nucleic acids from sub-optimal FFPE samples containing minimal amounts of tissue. Furthermore, the fixation process leads to the cross linking of nucleic acids and proteins resulting in highly fragmented nucleic acid species with amplimers around 100 bases. This can introduce sequence alterations and mono methylol addition of nucleic acids which can impact downstream PCR based assays [12–15]. A number of companies supply off the shelf kits optimised for the extraction of DNA and/or RNA from FFPE sections using varying amounts of tissue. Therefore, we carefully selected seven commercially available FFPE extraction kits with a broad range of properties to assess nucleic acid yield and the quality and purity of the extracted material.
Differing extraction methods were assessed: dual DNA/RNA extraction vs. specialised single extraction, manual silica column based extraction techniques vs. automated magnetic bead based methods and finally a comparison between extraction kits across a range of manufacturers. A number of papers have been published previously comparing the performance of manual FFPE extractions kits from different suppliers [16–18], with more recent studies focusing on comparisons between automated extraction methods and their manual counterpart in order to reduce the hands on time of laborious extraction processes [19–22]. However, the studies use different samples and methods for quantifying and assessing the quality of the extracted material that are not always comparable, making it challenging to cross compare disparate data sets. It is for this reason that we present a comprehensive analysis of seven selected kits using a single sample set. In addition, we provide a head-to-head comparison of commonly used platforms to aid decisions around how to extract, quantify, and assess the quality of nucleic acids.
Cell blocks were used to ensure sufficient sections could be generated to enable robust analysis of each extraction method; although tissue blocks were initially used, the material was rapidly exhausted thereby preventing all extraction kits from being evaluated across all variables using tissue from a single block. Cancer cell lines were cultured, fixed in formalin and embedded in paraffin, to represent the process of preserving clinical tissue whilst achieving a homogenous cell population with known expression of genes of interest for downstream evaluation. This ensured that variability in this assessment is solely due to differences in the extraction kits and not by fixation methods, times and heterogeneity of the tissue. An additional advantage of having a large quantity of cells embedded within a block, is that it allows each kit to be tested using optimal conditions, so significant differences between kits can be identified.
Formalin fixed paraffin embedded cancer cell line blocks
Human chronic myeloid leukaemia and breast cancer cell lines, used in this study, were obtained from the American Type Culture Collection (ATCC) and the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). Cells were grown in T225 tissue culture flasks in RPMI1640 media (phenol red free; Sigma) supplemented with 10% foetal calf serum and 1% L-Glutamine (Life Technologies Bethesda Research Laboratories Ltd) and maintained at 37 °C with 5% CO2. Each cell line was passaged up to 3 times and at approximately 80% confluence, the cells were fixed in 10% neutral buffered formalin for 24 h at room temperature before embedding in paraffin wax. Consecutive 5 μm sections were generated from each of the FFPE cell blocks using a standard microtome blade and fixed onto glass microscope slides. All FFPE cell blocks and 5 μm sections were stored at room temperature until nucleic acid extraction was performed.
Nucleic acid extraction
Summary of the seven off the shelf nucleic extraction kits evaluated in this study, including the key differences between each method
Input tissue amount
Level of automation
Proteinase K digestion
DNase I digestion
Geometric mean Yield (ng)
Up to 40 μm
15 min at 56 °C,
15 min at 80 °C
15 min at room temperature in the cell lysate
Arcturus Paradise plus RNA extraction and isolation
Up to 40 μm
5 h at 37 °C
20 min at room temperature on the column
Maxwell 16 LEV RNA FFPE Kit
Up to 10 μm
15 min at 56 °C,
60 min at 80 °C
15 min at room temperature in the cell lysate
AllPrep DNA/RNA FFPE
RNA & DNA
Up to 40 μm
RNA 30 μl DNA 50 μl
15 min at 56 °C
15 min at room temperature on the column
Arcturus PicoPure DNA extraction kit
1.5–2.0 μg tissue
Single tube extraction
24 h at 65 °C
(lyophilised before use)
QIAmp DNA FFPE tissue
Up to 80 μm
60 min at 56 °C,
60 min at 80 °C
Maxwell 16 FFPE Tissue LEV DNA Purification Kit
Up to 50 μm
Overnight at 70 °C
Summary of the FFPE cell pellet blocks used in the study
<6 months of age
>2 years of age
RNA extraction kits
DNA extraction kits
Quantitative and qualitative RNA assessment
RNA concentration was determined using the Qubit HS RNA assay (Life Technologies, catalogue number Q32852) on the Qubit 2.0 fluorometer. 1.0 μl of RNA in a total 200 μl volume of working solution was prepared. The fluorescence of unknown RNA samples was measured and converted to RNA concentration using a calibration curve generated from newly prepared RNA standards at 0 ng/μl and 10 ng/μl.
The extent of RNA fragmentation was assessed via capillary chip electrophoresis. RNA samples at a concentration ≥5 ng/μl were tested using RNA 6000 nano LabChips. The chips were run on the Agilent 2100 Bioanalyser according to the manufacturer’s instructions.
RNA concentration and purity were measured on the Nanodrop 2000 using 1 μl of RNA. The ratio between the absorbance at 260 nm and 280 nm was used to evaluate purity; we assumed ratios between 1.8 and 2.0 to be pure.
mRNA quality assessment
Multiplex quantitative 2-step reverse transcriptase PCR was performed to assess mRNA quality for the same sample set analysed on the Agilent. 7 μl of RNA at a concentration of 5 ng/μl (quantified using the Qubit 2.0 fluorometer) was used to generate cDNA in a 10 μl total extraction volume using the SuperScript VILO cDNA synthesis kit (Life Technologies, Catalogue Number 11754-050) followed by incubation at 25 °C for 10 min, 42 °C for 60 min and 85 °C for 5 min.
The qPCR assay was prepared in Roche 384 well PCR plates (product no. 04729749001) in a 10 μl reaction volume fired using an ECHO 525 non-contact micro dispenser (LabCyte). Three primer probe pairs were used in each assay against the following housekeeping genes: RPLP0 (CY5), RPL19 (FAM) and ACTB (CYAN500) using three unique fluorophores compatible with the Roche LightCycler480 PCR machine. 0.5 μl of cDNA at 3.5 ng/μl (within the linear range of the assay, Additional file 1: Figure S1) was run in duplicate alongside negative controls lacking the cDNA template on the Roche LightCycler480 PCR machine with 1 pre incubation cycle at 95 °C for 10 min followed by 45 amplification cycles at 95 °C for 10 s, 60 °C for 30 s, 72 °C for 1 s before cooling to 40 °C for 30 s. Data was analysed using the LightCycler 480 software (version 22.214.171.124) under the “Abs quant/ 2nd Derivative Max analyses” programme. This software calculated the average Cq per gene for sample duplicates; this was then used to calculate the average across the 3 house keeping genes and this value was used to compare mRNA quality obtained using the different extraction kits, lower Cq values indicated higher quality mRNA.
Quantitative and qualitative DNA assessment
For DNA to be used in downstream genomic techniques, for example NGS, quality and quantity of amplifiable DNA needs to be assessed. Qubit and Nanodrop will not generate this information so Quantitative PCR was performed for DNA quantification and quality assessment, using the hgDNA Quantification and QC kit from Kapa Biosystems (KK4960) as per the manufacturer’s instructions. Two amplicons from a conserved single copy DNA locus were generated, one 41 bp in length and one 129 bp in length. Amplification of the 41 bp target was used for quantitation of amplifiable DNA. This was compared to data generated using the 129 bp assay, to ensure valid conclusion for the Qiagen extraction kits that are less able to retain fragments <100 bp. The 129 bp assay was used to assess DNA quality, since poor DNA quality influences the ability to amplify longer DNA fragments. The relative quality of DNA samples was calculated by normalising the concentration obtained using the 129 bp assay against the concentration obtained from the 41 bp assay, which generates a Q-ratio value between 0 and 1 (0 represents highly fragmented DNA whilst 1 represents high quality DNA). Both assays were performed on the Roche LightCycler480 PCR machine in a 3.0 μl reaction volume. Data was analysed using the Roche LC480 software using the “Abs quant/ 2nd Derivative Max analyses” programme. This software calculated the average Cq value of duplicates, the standard deviation and DNA concentrations were generated from a standard curve.
Graphical representations were generated using TIBCO Spotfire software version 5.0 and Microsoft Office Excel 2007. Data was represented as the geometric mean with corresponding geometric standard error as the data was found to have an asymmetrical distribution. The statistical significance of the data was assessed using two sample unequal variance t tests as preliminary analysis was performed in the absence of an appropriate rationale as to the direction of the relationship. In the results and discussion, we refer to P-values <0.05 as significant. The coefficient of variation was calculated for correlation plots to assess the fit of simple linear regressions.
Results and discussion
Total RNA yield
Both Qiagen RNA extraction kits significantly outperformed the Artcturus paradise plus FFPE RNA isolation kit and the automated Maxwell 16 LEV RNA FFPE Kit. The RNeasy FFPE Kit gave geometric mean RNA yields 166 ng greater than the Maxwell 16 and 200 ng greater than the Artcturus paradise plus FFPE RNA isolation kit. The AllPrep gave geometric mean RNA yields 36 ng greater than the Maxwell 16 and 70 ng greater than the Arcturus paradise plus FFPE RNA isolation kit. Multiple factors may contribute to the lower RNA yields obtained using the Artcturus paradise plus FFPE RNA isolation kit. Firstly, the lower temperature used during tissue digestion may less efficiently reverse formaldehyde-induced cross links between nucleic acids and proteins, previous studies have reported heating tissues at higher temperatures (>50 °C) as being more efficient [13, 23]. Also the lengthy 5 h digestion time may not be necessary as previous papers have published that no more than 3 h is required . Finally, this method uses less than half the elution volume of the two Qiagen kits potentially compromising the efficiency of the elution buffer to cover the silica membrane which could contribute to the lower RNA yield observed. Despite the lower yield, the Artcturus kit gave the highest RNA concentration which may be desirable for some studies (Table 1).
The Maxwell 16 RNA extraction is the only method that does not involve deparaffinisation; we hypothesise that excess paraffin may reduce the efficiency of cell lysis, since some studies have shown deparaffinisation to be a key pre-treatment step . In addition, this protocol uses paramagnetic beads for purification as opposed to silica columns. The speed at which the beads are mixed with the cell lysate has been shown to have a strong influence on the efficiency of nucleic acid binding with faster mixing speeds being linked to higher RNA yields. Furthermore, the process of transferring the beads through a series of wash buffers can result in the loss of beads which will negatively impact yield. Although the Maxwell 16 automated the binding, washing, and elution steps, up-front sample preparation was required which took approximately 2 h before loading the lysate onto the machine. In addition, it limited the number of samples processed per run to 16 as opposed to batches of 24, which we feel can comfortably be processed using any of the manual extraction kits. This shows the automated RNA extraction on the Maxwell 16 provides no increase in throughput, and we saw no advantage in terms of yield when using this bead based purification method for RNA extraction over silica columns.
Fluorescence vs. absorbance for RNA quantification
Each of the extracted RNA samples were quantified using both the Qubit RNA HS fluorescence based assay and the Nanodrop 2000 spectrophotometer (for 5 samples there was insufficient sample remaining to run on the Nanodrop). The Nanodrop also generated A260/A280 ratios as a means of assessing purity. All four RNA extraction kits gave mean ratios ≥1.8 indicating good purity.
Nanodrop consistently underestimated the RNA concentration due to the lower sensitivity of the assay (Additional file 2: Figure S2). The limit of detection of the Nanodrop 2000 is 2 ng/μl whereas for the Qubit it is 0.25 ng/μl; 37% of the samples extracted using the Maxwell 16 gave RNA concentrations <2 ng/μl but greater that 0.25 ng/μl and therefore fell within the background noise of the Nanodrop assay but not the Qubit. For these reasons we chose to use RNA concentration readings generated from the Qubit in all subsequent analyses.
Effect of tissue input on RNA concentration/yield using a fixed elution volume
Total nucleic acid yield and concentration are influenced by the elution volume and the amount of tissue processed. We investigated the effect of varying the amount of tissue on nucleic acid concentration and yield by processing 5 μm, 10 μm, 15 μm, 20 μm and 25 μm of tissue.
Therefore, our data shows that combining tissue sections offers no advantage in terms of yield when compared to extracting sections individually, but does provide a method of increasing RNA concentration although in a non-linear fashion.
Effect of elution volume on RNA concentration/yield using a fixed amount of tissue
In previous experiments the RNeasy FFPE kit gave superior RNA yields, therefore we investigated the relationship between RNA yield/concentration and elution volume. 12 μl, 20 μl, 30 μl 40 μl and 50 μl elution volumes were tested using 5 μm of tissue from four different FFPE cancer cell line blocks.
Results showed a 20 μl elution volume was optimal and resulted in high RNA yields and RNA concentrations (Additional file 3: Figure S3). However, we acknowledge that the amount of RNA bound to the column will also have an impact on elution efficiency; samples with higher amounts of RNA may require larger elution volumes.
RNA quality assessment: Agilent vs RT-PCR
RNA samples with a concentration ≥5 ng/μl (limit of detection of the Agilent nano assay), obtained using four different extraction kits, were analysed for quality using the Agilent RNA 6000 nano assay. The assigned RNA integrity number (RIN) values were compared to the in house multiplex RT-PCR assay as an assessment of amplifiable RNA. The RNA samples fell into two groups, those obtained from FFPE cell line pellets generated <6 months prior to commencing the study and those obtained from FFPE cell line pellets generated >2 years prior to commencing the study.
To allow for a more sensitive, quantitative assessment of RNA quality isolated from FFPE samples, we developed a multiplex PCR assay in house targeting 3 housekeeping genes ActB, RPL19 and RPLP0 to asses RNA quality based on the ability to amplify targets. The multiplex PCR assay showed good concordance with single-plex PCR assays for each housekeeping gene (Additional file 4: Figure S4) and little intra-assay variation was seen, demonstrated by the strong correlation (R2 value of 0.985) between technical replicates (Additional file 5: Figure S5). Only RNA samples that were at a concentration ≥5 ng/μl were analysed in this assay.
The multiplex PCR assay was able to demonstrate statistically significant differences in the quality of the RNA released from the different age FFPE blocks (Fig. 4b). Across all the extraction kits, lower quality RNA was obtained from the blocks fixed between 2 and 3 years ago than those fixed <6 months ago. Furthermore, differences in the quality of the RNA released from different RNA extraction kits were also found to be statistically significant. The Maxwell 16 gave significantly higher quality RNA (lowest mean Cq value) than the three other extraction methods, although only 50% of samples were >5 ng/μl indicating a compromise in yield. The mean Cq value for the Maxwell 16 was 25.9, 2.4 Cq lower than the Arcturus Paradise Plus FFPE RNA isolation kit, 1.6 Cq lower than the RNeasy and 2.0 Cq lower than the Dual extraction kit. The Maxwell 16 is the only method which uses paramagnetic beads and the lack of mechanical force applied through centrifugation in this extraction technique may account for the higher quality of the RNA released. No significant differences in the mean Cq values between the two Qiagen extraction kits were seen. This data demonstrates the in house multiplex PCR assay is a much more sensitive approach, allowing multiple measures of RNA quality from minimal amounts of sample.
Total amplifiable DNA recovery
A total of 120 DNA samples from 6 different FFPE cell line pellets were obtained using four different DNA extraction kits. 5–25 μm of tissue from each block was processed using each method. DNA concentration was quantified based on the amplification of a 41 bp DNA target from a conserved single copy DNA locus. Due to Qiagen kits not retaining products <100 bp, the DNA concentration was also quantified using amplication of a 129 bp DNA target.
Based on this data, the Arcturus PicoPure DNA extraction kit demonstrated superior performance in terms of DNA yield from FFPE tissue.
Effect of tissue input on DNA concentration/yield using a fixed elution volume
Each DNA extraction method was tested using 5 μm, 10 μm, 15 μm, 20 μm and 25 μm of tissue from 6 different FFPE cancer cell line pellets. The relationship between DNA concentration, quantified by qPCR as above, and amount of tissue processed was investigated.
Overall results showed that extraction from 20 μm tissue sections gave optimal yields across all the kits (Fig. 2).
DNA quality assessment
The quality of 120 DNA samples extracted from different age FFPE blocks using four different extraction kits was assessed using two qPCR assays targeting a 41 bp amplicon and a 129b amplicon from a conserved single copy DNA locus. The ratio between the concentration of the 129 bp amplicon and 41 bp amplicon (Q ratio) was used as a measure of DNA quality as fragmentation will impact the amplification of the longer DNA target, resulting in a lower ratio. However, running this assay in the recommended 20 μl reaction volume with sample duplicates, limits the ability to assess the quality of scarce clinical samples, so the assay was miniaturised to a 3.0 μl reaction volume.
Silica column and bead based extractions rely on the DNA binding ability of a solid phase. The columns supplied in the Qiagen single and dual DNA extraction kits are only able to bind DNA molecules ≥100 base pairs in length, resulting in the loss of the 41 bp fragment and selective enrichment of DNA fragments ≥100 base pairs. This leads to an over estimation of the ratio between the 129 bp target and the 41 bp target, so caution when analysing this data is required. In contrast, the Picopure DNA extraction kit is not reliant on the binding of DNA molecules to a solid phase and therefore all DNA fragments are retained, giving a more realistic representation of the quality of the DNA released from FFPE samples.
Data showed the miniaturised KAPA assay can be used to assess quantity and quality of DNA extracted without sacrificing large amounts of DNA (Fig. 7).
Previous publications have compared the performance of manual FFPE extraction kits [16–18] and automated and manual kit counterparts [19–22], but different samples and methods for quantifying and assessing quality makes comparisons challenging. We have presented a comprehensive analysis of seven selected kits with a single sample set and compared directly platforms to assess quantity and quality of nucleic acids.
This study demonstrates that a number of factors influence the quantity and quality of nucleic acids obtained from FFPE samples, including the age of the samples, the extraction method, the amount of tissue processed and the elution volume. Following the manufacturer’s instructions, the data showed that no single protocol will consistently release high yields of high quality nucleic acids across all sample types.
Perhaps not surprisingly, our data shows that superior performance in high yields will often result in a compromise in concentration and/or quality. Therefore, downstream applications and nature of the samples to be tested should be given consideration up front before choosing the extraction method to apply.
Although FFPE cell line material was used in this study to remove sample heterogeneity, and supply issues, Qiagen RNeasy FFPE kit has been successfully utilised on clinical FFPE samples following this study . Indicating that conclusions from this study are applicable to clinical FFPE tissue samples.
To potentially improve the performance of the kits tested in this study a wider validation study would need to be conducted, to test variables such as; method of deparaffinisation (deparaffinisation buffers vs. xylene), lysis volume and incubation time, column vs. cell lysate DNase digestion (for RNA isolation), and testing reagents such as Proteinase K from a lyophilised stock rather than a readymade solution.
This study successfully highlighted the key benefits of each extraction method tested, following the manufacturer’s protocols, with the aim to aid scientists in choosing a particular method dependent on individual goals. We provide a flow diagram to help question the properties that are most important to the success of studies with additional information around the most accurate and efficient methods to quantify and QC the extracted material (Fig. 2).
Formalin fixed paraffin embedded
AstraZeneca funded the study and AstraZeneca employees. This experimental design, analysis and interpretation along with authorship of the manuscript was also funded by AstraZeneca.
Availability of data and materials
All data generated or analysed during this study are included in this published article [and its Additional files 1, 2, 3, 4, 5 and 6]. Raw data is not publically available but can be shared from the corresponding author on reasonable request.
CS generated and analysed data, and wrote manuscript. AS performed statistical analyses of the data, and contributed to writing the manuscript. GBM, JCB, LH, and EJ provided key technical and scientific input, and all were major contributors in writing the manuscript. All authors read and approved the final manuscript.
CS has no competing interests. GBM, AS, JCB and EAH and EVJ are employees and shareholders at AZ and have no other competing interests.
Consent for publication
Ethics approval and consent to participate
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