The goal of this video is to demonstrate how to perform automated DNA extraction from formalin-fixed paraffin-embedded (FFPE) reference standard cell lines and digital droplet PCR (ddPCR) analysis to detect rare mutations in a clinical setting. Detecting mutations in FFPE samples demonstrates the clinical utility of ddPCR in FFPE samples.
ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.
The accumulation of genetic mutations in key regulatory genes alters normal cell programing like cell proliferation, differentiation, and survival, leading to cancer1. The RAS-RAF-MAP kinase pathway mediates cellular responses to growth signals. Oncogenic BRAF mutations can result from driver mutations in the BRAF gene, which may cause the BRAF oncoprotein to become overactive2. Mutations in the BRAF gene also result in overactive downstream signaling via MEK and ERK3, which, in turn, leads to excessive cell growth and proliferation independently of growth factor-mediated regulation4-6.
Several tools are available for DNA mutation profiling, such as quantitative real-time BRAF V600E mutations in formalin-fixed, paraffin-embedded (FFPE) reference standard cell lines by ddPCR. ddPCR is an PCR-based method for absolute quantification offering higher accuracy compared to conventional quantitative real-time PCR (qPCR)7,8. ddPCR also provides higher resolving power and accuracy for the detection of rare mutations in DNA templates, enabling more informative cancer research and diagnosis9. Additional advantages of ddPCR over conventional qPCR include its enhanced sensitivity and accuracy when studying low template copy numbers10-12. Herein, a protocol for automatically extracting DNA from FFPE reference standard cell lines, followed by determining the presence or absence of BRAF V600E mutations by ddPCR is demonstrated. The usage of software for data analysis and a graphical representation of the results are also described. The entire procedure is relatively simple and totally depends on the number of samples to be profiled and the number of conventional PCR and ddPCR machines available.
The following protocol describes standard procedures for BRAF V600E-positive FFPE reference standard cell lines (HD598, HD593, HD617, HD273 and wildtype (WT)) is performed in a fully automated instrument using the Tissue Preparation System (TPS) protocol. Subsequently, isolated DNA samples are analyzed for the presence of BRAF V600E mutations using ddPCR system. Targeted mutation analysis is performed after all samples have been profiled and the data has been loaded into the data analysis software. Depending on the number of samples/groups studied, data analysis may require from one to several hours. The experimental component of the methodology requires accuracy in handling DNA and pipetting into 96 well plates, while data analysis is performed using software.
1. DNA Extraction from FFPE Reference Standard Cell Lines
Note: For this procedure, DNA extraction was performed from FFPE reference standard cell lines (HD598, HD593, HD617, HD273 and wildtype (WT)) using the FFPE Tissue DNA isolation kit as described in the protocol below. Automated DNA extraction was achieved by following the manufacturer's instructions for total DNA isolation.
1.2 TPS protocol
Note: The volumes shown in Table 1 correspond to the minimum required to process 48 samples, and the procedure shown is in accordance with the TPS guidelines. Before starting the experiment settle down the FFPE samples in the e-tube by centrifugation at 600 x g, to avoid loss of samples during the automated program.
2. DNA Mutation Profiling: ddPCR Protocol
Note: The protocol for DNA mutation profiling consists of 3 major steps:1) Droplet generation, 2) Conventional PCR amplification, 3) Droplet reading and 4) DNA mutation profiling.
2.1. Droplet generation
Note: ddPCR supermix is recommended for ddPCR, as this mix contains reagents required for droplet generation.
2.2. Preparation for PCR
2.3 Droplet reading (as per the manufacturer’s recommended protocol)
Note: Following PCR amplification of the nucleic acid target in the droplets, the droplet reader instrument analyzes each droplet individually using a 2-color detection system13. We typically set to detect FAM and VIC reporter fluorophores.
2.4 DNA mutation profiling (as per the manufacturer’s recommended protocol)
Note: PCR-positive and PCR-negative droplets are counted to provide absolute quantification of target BRAF V600E DNA mutations in digital form, using data analysis software.
For our ddPCR analysis, we studied the BRAF V600E mutation FFPE reference standard cell lines. The droplet reader connects to a laptop computer running data analysis software. Each individual droplet is defined on the basis of fluorescent amplitude as being either positive or negative. The software provided by manufacturer also allows a user-defined cutoff to be entered to define the threshold between the positive and negative droplets. The number of positive and negative droplets in a sample is used to calculate the concentration of target in terms of copies/µl.
Fluorescence was detected and processed into a two-dimensional scatter plot display, custom software was used to draw appropriate gates for each droplet endpoint cluster, and the number of droplets within each gate was counted. As shown in Figure 3A, droplets represented by blue dots (FAM fluorescence signal) above the cut-off line for all samples (pink line) were positive for mutated BRAF V600E. Droplets represented by blue dots in the BRAF WT (NTC; Lane 2) sample could be due to a non-specific signal (false positive). False positive signals (BRAF WT) were normalized with other mutation samples. As shown in Figure 3B, WT BRAF droplets are represented by green dots (VIC fluorescence signal). In both plots, the grey dots at the bottom are considered as the fluorescence background. The overall mutant allele frequencies were calculated using the data shown in Figure 3C, based on the relative percentages of WT BRAF and BRAF V600E templates detected. Obtained ddPCR results contain the droplet event counts and calculated wild-type and mutant DNA molecule counts for the BRAF V600E (50%, 10%, 5%, 1%, 0.5% , 0.1% and 0.05%) samples calculated by using the below mentioned formula .
% of Mutant frequency = (Mutant copy / (Wildtype + Mutant copy)) x 100
Accordingly, BRAF V600E mutations were identified and verified with reference standard (BRAF WT). Defined BRAF V600E mutation allelic frequencies of 50%, 10%, 5%, 1%, 0.5%, 0.1% and 0.05% were used to test the sensitivity and reproducibility of the ddPCR system. From our analysis with known sample concentrations, we confirmed that ddPCR is able to detect as low as 0.05% of BRAF V600E mutation. The detection of false positive mutant count in NTC or WT might possibly be due to non-specific probe hydrolysis as reported earlier 14. Detection of more than two copies in a sample has been considered as positive in tumor tissue 15.
Figure 1. Schematic representation describing reagent and sample loading in preparation for automated DNA extraction instrument. Place the samples in a carrier racks and dispense the reagents into corresponding troughs as mentioned. Employing automated TPS protocol that supports multiple sample types, delivers accurate, and reliable results with maximum productivity. Re-printed with permission from Siemens Healthcare Diagnostics. (courtesy of Siemens Healthcare Diagnostics).
Figure 2. Schematic representation of the Tissue Preparation System Workflow for automated DNA extraction. Fully automated DNA isolation procedure for FFPE tissues sections including negative selection steps of paraffin, tissue debris removal and positive selection steps of binding and elution are shown. Re-printed with permission from Siemens Healthcare Diagnostics. (courtesy of Siemens Healthcare Diagnostics).
Figure 3. Use of the ddPCR system for precise quantification of the BRAF V600E mutation in FFPE reference standard cell line samples. (A, B) Visualization of positive fluorescence amplitudes in 1D plots (1dot -1droplet). Blue dots (A, FAM positive) represent mutant BRAF V600E-positive droplets, while green dots (B, VIC positive) represent WT BRAF-positive droplets. This determination enables precise mutation quantification in FFPE reference standard cell lines. The pink line is the discrimination threshold between positive and negative signals of the droplets. (C) The fractional abundance plot shows blue markers that indicate the concentration (copies/µl) of BRAF V600E mutation, and the green markers indicate the concentration (copies/µl) of BRAF (WT). All error bars generated by data analysis software represent the 95% confidence interval.
Reagents | Volume (ml) |
Lysis Buffer | 106 ml |
Wash Buffer 1 | 101 ml |
Wash Buffer 2 | 72 ml |
Wash Buffer 3 | 106 ml |
Elution Buffer | 19 ml |
Magnetic beads | 8 ml |
FFPE buffer | 15 ml |
Proteinase K | 3.3 ml |
Table 1. Total volume of reagents (TPS kit) required for DNA extraction with 48 samples.
Cycling Step | Temperature | Time | # Cycles |
Enzyme activation | 95 °C | 10 min | 1 |
Denaturation | 94 °C | 30 sec | 40 |
Annealing/extension | 60 °C | 1 min * | |
Hold | 98 °C | 10 min | 1 |
Hold | 4 °C | Forever | 1 |
* Adjust ramp rate settings to 2-2.5 °C/sec. Use a heated lid set to 105 °C and set the sample volume to 40 μl |
Table 2. Conventional PCR thermocycling conditions
Here, we highlight the applicability of ddPCR and DNA isolation from FFPE reference standard cell line samples for a specific gene mutation assessment. In this study, TPS automated DNA isolation method is used which can be readily adapted, automated, and can accommodate up to 48 different samples simultaneously, allowing for larger scale experiments and lower variability. One of the limitations of the DNA isolation in the present work is that every FFPE sample is unique, and will vary one another in surface contaminants, microbial flora, and/or human genetic backgrounds. In general, extracted DNA quality and quantity and the success of whole genomic DNA amplification are dependant upon various parameters before, during and after extraction. These include, type and amount of tissue, type of fixative used for tissue preservation, duration of fixation, age of the paraffin block and storage conditions, as well as the length of the desired DNA segment to be analysed16. Removal of paraffin from the tissue is the most critical step for successful extraction as undissolved paraffin leads to poor sample quality. During the droplet generation, care must be taken to prevent bubble formation – this is another critical step for successful mutation detection. Considering the sample to sample variation that might arise between sample populations and based on the motive of the experiment, certain modifications in the procedure might be required to obtain desired result.
Another advantage is that DNA isolation and ddPCR is conducted using automated systems in this protocol, and hence there is negligible error and user intervention required is very minimal. Isolating whole-genome-amplified DNA from paraffin-embedded tissue/cells was obtained by using TPS system. One of the drawbacks in using automated DNA isolation system is that, it is not cost efficient to use small number of samples. Instead of this automated step, other standard DNA isolation procedure could also be performed for limited samples
A recent study stated that using droplet digital PCR (ddPCR) is able to determine the relative copy number of specific genomic loci even in the presence of intermingled normal tissue obtained from FFPE tissues. By using a control dilution series, Nadauld, L. et al. determined the limits of detection (LOD) for the ddPCR assay and reported its improved sensitivity on minimal amounts of DNA compared to standard real-time PCR17. Here, FFPE reference standard cell lines are used to demonstrate the mutation detection capability of the ddPCR system. The ddPCR system results indicated the possibility to detect rare mutation allelic frequencies down to 0.05% mutation. Collectively, these data indicate that the ddPCR system also enables quantitative analysis of the percentages of various mutant alleles and relative differences in heterozygous clinical tumor samples. Large number of FFPE samples can be analyzed for specific gene mutations simultaneously and this is an optimal technique for population wide genetic studies.
Finally, it should be taken into account that the mutation frequencies are represented here are absolute quantification and should not be considered as relative value of mutation rate or frequency. ddPCR readout provides absolute quantification of target DNA mutation. These values can be used for validating mutation frequencies of samples prepared under the same condition, and sequenced over the same region. However, these absolute values are reproducible and can be used for quantitative comparison of mutation distribution and frequency when optimal parameters are controlled. In conclusion, ddPCR has recently emerged as a robust tool that gives absolute quantitation of nucleic acids in FFPE and biopsy samples and also can be duplexed with reference assays for determination of either normalized transcript concentrations or DNA copy number.
The authors have nothing to disclose.
This research was supported by the R&D Program for the Society of the National Research Foundation (NRF), funded by the Ministry of Science, ICT & Future Planning (Grant No. 2013M3C8A1075908).
Hamilton MICROLAB STARlet IVD instrument | Siemens | 10701001 | Automated DNA isolation instrument |
QX200 Droplet Generator | Bio-Rad | 772BR1119 | |
QX200 Droplet Reader | Bio-Rad | 771BR1497 | |
Conventional PCR machine capable of ramp-time adjustment | 621BR17718 | ||
PX1 PCR plate sealer | Bio-Rad | 770BR1575 | |
QuantaSoft software | Bio-Rad | ||
DNA isolation kit | |||
VERSANT Tissue Preparation Reagents Box 1 | Siemens | 10632398 | |
VERSANT Tissue Preparation Reagents Box 1 | Siemens | 10632399 | |
CO-RE tips | Siemens | ||
ddPCR mutation analysis | |||
ddPCR Supermix | Bio-Rad | BR186-3010 | 2X concentration |
DG8 cartridge | Bio-Rad | BR186-4008 | |
Droplet Generator oil | Bio-Rad | BR-186-3005 | |
Gasket | Bio-Rad | BR186-3006 | |
Droplet reader oil | Bio-Rad | BR-186-3004 |