Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions

The Qubit fluorometer is a DNA quantification device based on the fluorescence intensity of fluorescent dye binding to double-stranded DNA (dsDNA). Qubit is generally considered useful for checking DNA quality before next-generation sequencing because it measures intact dsDNA. To examine the most accurate and suitable methods for quantifying DNA for quality assessment, we compared three quantification methods: NanoDrop, which measures UV absorbance; Qubit; and quantitative PCR (qPCR), which measures the abundance of a target gene. For the comparison, we used three types of DNA: 1) DNA extracted from fresh frozen liver tissues (Frozen-DNA); 2) DNA extracted from formalin-fixed, paraffin-embedded liver tissues comparable to those used for Frozen-DNA (FFPE-DNA); and 3) DNA extracted from the remaining fractions after RNA extraction with Trizol reagent (Trizol-DNA). These DNAs were serially diluted with distilled water and measured using three quantification methods. For Frozen-DNA, the Qubit values were not proportional to the dilution ratio, in contrast with the NanoDrop and qPCR values. This non-proportional decrease in Qubit values was dependent on a lower salt concentration, and over 1 mM NaCl in the DNA solution was required for the Qubit measurement. For FFPE-DNA, the Qubit values were proportional to the dilution ratio and were lower than the NanoDrop values. However, electrophoresis revealed that qPCR reflected the degree of DNA fragmentation more accurately than Qubit. Thus, qPCR is superior to Qubit for checking the quality of FFPE-DNA. For Trizol-DNA, the Qubit values were proportional to the dilution ratio and were consistently lower than the NanoDrop values, similar to FFPE-DNA. However, the qPCR values were higher than the NanoDrop values. Electrophoresis with SYBR Green I and single-stranded DNA (ssDNA) quantification demonstrated that Trizol-DNA consisted mostly of non-fragmented ssDNA. Therefore, Qubit is not always the most accurate method for quantifying DNA available for PCR.     

Comparison of Pre-Analytical FFPE Sample Preparation Methods and Their Impact on Massively Parallel Sequencing in Routine Diagnostics

Over the last years, massively parallel sequencing has rapidly evolved and has now transitioned into molecular pathology routine laboratories. It is an attractive platform for analysing multiple genes at the same time with very little input material. Therefore, the need for high quality DNA obtained from automated DNA extraction systems has increased, especially to those laboratories which are dealing with formalin-fixed paraffin-embedded (FFPE) material and high sample throughput. This study evaluated five automated FFPE DNA extraction systems as well as five DNA quantification systems using the three most common techniques, UV spectrophotometry, fluorescent dye-based quantification and quantitative PCR, on 26 FFPE tissue samples. Additionally, the effects on downstream applications were analysed to find the most suitable pre-analytical methods for massively parallel sequencing in routine diagnostics. The results revealed that the Maxwell 16 from Promega (Mannheim, Germany) seems to be the superior system for DNA extraction from FFPE material. The extracts had a 1.3–24.6-fold higher DNA concentration in comparison to the other extraction systems, a higher quality and were most suitable for downstream applications. The comparison of the five quantification methods showed intermethod variations but all methods could be used to estimate the right amount for PCR amplification and for massively parallel sequencing. Interestingly, the best results in massively parallel sequencing were obtained with a DNA input of 15 ng determined by the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). No difference could be detected in mutation analysis based on the results of the quantification methods. These findings emphasise, that it is particularly important to choose the most reliable and constant DNA extraction system, especially when using small biopsies and low elution volumes, and that all common DNA quantification techniques can be used for downstream applications like massively parallel sequencing.     

Improved microRNA quantification in total RNA from clinical samples

microRNAs are small regulatory RNAs that are currently emerging as new biomarkers for cancer and other diseases. In order for biomarkers to be useful in clinical settings, they should be accurately and reliably detected in clinical samples such as formalin fixed paraffin embedded (FFPE) sections and blood serum or plasma. These types of samples represent a challenge in terms of microRNA quantification. A newly developed method for microRNA qPCR using Locked Nucleic Acid (LNA?)-enhanced primers enables accurate and reproducible quantification of microRNAs in scarce clinical samples. Here we show that LNA?-based microRNA qPCR enables biomarker screening using very low amounts of total RNA from FFPE samples and the results are compared to microarray analysis data. We also present evidence that the addition of a small carrier RNA prior to total RNA extraction, improves microRNA quantification in blood plasma and laser capture microdissected (LCM) sections of FFPE samples.     

qPCR-based mitochondrial DNA quantification: influence of template DNA fragmentation on accuracy

Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA (λnDNA) and mtDNA (λmtDNA) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two genomes is demonstrated and which evaluates systematically the impact of DNA degradation on quantification of mtDNA copy number.     

A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA

Background

Formalin fixed paraffin embedded (FFPE) tumor samples are a major source of DNA from patients in cancer research. However, FFPE is a challenging material to work with due to macromolecular fragmentation and nucleic acid crosslinking. FFPE tissue particularly possesses challenges for methylation analysis and for preparing sequencing-based libraries relying on bisulfite conversion. Successful bisulfite conversion is a key requirement for sequencing-based methylation analysis.

Methods

Here we describe a complete and streamlined workflow for preparing next generation sequencing libraries for methylation analysis from FFPE tissues. This includes, counting cells from FFPE blocks and extracting DNA from FFPE slides, testing bisulfite conversion efficiency with a polymerase chain reaction (PCR) based test, preparing reduced representation bisulfite sequencing libraries and massively parallel sequencing.

Results

The main features and advantages of this protocol are:

• An optimized method for extracting good quality DNA from FFPE tissues.
• An efficient bisulfite conversion and next generation sequencing library preparation protocol that uses 50 ng DNA from FFPE tissue.
• Incorporation of a PCR-based test to assess bisulfite conversion efficiency prior to sequencing.

Conclusions

We provide a complete workflow and an integrated protocol for performing DNA methylation analysis at the genome-scale and we believe this will facilitate clinical epigenetic research that involves the use of FFPE tissue.

     

Amplicon Sequencing of Colorectal Cancer: Variant Calling in Frozen and Formalin-Fixed Samples

Next generation sequencing (NGS) is an emerging technology becoming relevant for genotyping of clinical samples. Here, we assessed the stability of amplicon sequencing from formalin-fixed paraffin-embedded (FFPE) and paired frozen samples from colorectal cancer metastases with different analysis pipelines. 212 amplicon regions in 48 cancer related genes were sequenced with Illumina MiSeq using DNA isolated from resection specimens from 17 patients with colorectal cancer liver metastases. From ten of these patients, paired fresh frozen and routinely processed FFPE tissue was available for comparative study. Sample quality of FFPE tissues was determined by the amount of amplifiable DNA using qPCR, sequencing libraries were evaluated using Bioanalyzer. Three bioinformatic pipelines were compared for analysis of amplicon sequencing data. Selected hot spot mutations were reviewed using Sanger sequencing. In the sequenced samples from 16 patients, 29 non-synonymous coding mutations were identified in eleven genes. Most frequent were mutations in TP53 (10), APC (7), PIK3CA (3) and KRAS (2). A high concordance of FFPE and paired frozen tissue samples was observed in ten matched samples, revealing 21 identical mutation calls and only two mutations differing. Comparison of these results with two other commonly used variant calling tools, however, showed high discrepancies. Hence, amplicon sequencing can potentially be used to identify hot spot mutations in colorectal cancer metastases in frozen and FFPE tissue. However, remarkable differences exist among results of different variant calling tools, which are not only related to DNA sample quality. Our study highlights the need for standardization and benchmarking of variant calling pipelines, which will be required for translational and clinical applications.     

Validation of extraction methods for total RNA and miRNA from bovine blood prior to quantitative gene expression analyses

The benefit and precision of blood diagnosis by quantitative real-time PCR (qPCR) is limited by sampling procedures and RNA extraction methods. We have compared five different RNA extraction protocols from bovine blood regarding RNA and miRNA yield, quality, and most reproducible data in the qRT-PCR with the lowest point of quantification. Convincing results in terms of highest quantity, quality, and best performance for mRNA qPCR were obtained by leukocyte extraction following blood lysis as well as extraction of PAXgene stabilized blood. The best microRNA qPCR results were obtained for samples extracted by the leukocyte extraction method.     

Validation of DNA methylation profiling in formalin-fixed paraffin-embedded samples using the Infinium HumanMethylation450 Microarray

A formalin-fixed paraffin-embedded (FFPE) sample usually yields highly degraded DNA, which limits the use of techniques requiring high-quality DNA, such as Infinium Methylation microarrays. To overcome this restriction, we have applied an FFPE restoration procedure consisting of DNA repair and ligation processes in a set of paired fresh-frozen (FF) and FFPE samples. We validated the FFPE results in comparison with matched FF samples, enabling us to use FFPE samples on the Infinium HumanMethylation450 Methylation array.     

Validation of DNA methylation profiling in formalin-fixed paraffin-embedded samples using the Infinium HumanMethylation450 Microarray

A formalin-fixed paraffin-embedded (FFPE) sample usually yields highly degraded DNA, which limits the use of techniques requiring high-quality DNA, such as Infinium Methylation microarrays. To overcome this restriction, we have applied an FFPE restoration procedure consisting of DNA repair and ligation processes in a set of paired fresh-frozen (FF) and FFPE samples. We validated the FFPE results in comparison with matched FF samples, enabling us to use FFPE samples on the Infinium HumanMethylation450 Methylation array.     

Droplet digital PCR is an accurate method to assess methylation status on FFPE samples

Most tissue samples available for cancer research are archived as formalin-fixed paraffin-embedded (FFPE) samples. However, the fixation process and the long storage duration lead to DNA fragmentation and hinder epigenome analysis. The use of droplet digital PCR (ddPCR) to detect DNA methylation has recently emerged. In this study, we compare an optimized ddPCR assay with a conventional qPCR assay by targeting a dilution series of control DNA. In addition, we compare the ddPCR technology with results from Infinium arrays targeting two separate CpG sites on a set of colon adenoma FFPE samples. Our data demonstrate that qPCR and ddPCR assess methylation status equally well on dilution controls with a high DNA input. However, the methylation detection on low-input samples is more accurate using ddPCR. The proposed primer design (methylation-independent primers with amplification of solely the converted DNA target) will allow for methylation detection, independent of bisulfite conversion efficiency. Those data show that ddPCR can be used for methylation analysis on FFPE samples with a wide range of DNA input and that the precision of the assay depends largely on the total amount of amplifiable DNA fragments. Due to accessibility of the ddPCR technology and its accuracy on high- as well as low-DNA input samples, we propose the use of this approach for studies involving degraded FFPE samples.     

Performance of Droplet Digital PCR in Non-Invasive Fetal RHD Genotyping - Comparison with a Routine Real-Time PCR Based Approach

Detection and characterization of circulating cell-free fetal DNA (cffDNA) from maternal circulation requires an extremely sensitive and precise method due to very low cffDNA concentration. In our study, droplet digital PCR (ddPCR) was implemented for fetal RHD genotyping from maternal plasma to compare this new quantification alternative with real-time PCR (qPCR) as a golden standard for quantitative analysis of cffDNA. In the first stage of study, a DNA quantification standard was used. Clinical samples, including 10 non-pregnant and 35 pregnant women, were analyzed as a next step. Both methods’ performance parameters—standard curve linearity, detection limit and measurement precision—were evaluated. ddPCR in comparison with qPCR has demonstrated sufficient sensitivity for analysing of cffDNA and determination of fetal RhD status from maternal circulation, results of both methods strongly correlated. Despite the more demanding workflow, ddPCR was found to be slightly more precise technology, as evaluated using quantitative standard. Regarding the clinical samples, the precision of both methods equalized with decreasing concentrations of tested DNA samples. In case of cffDNA with very low concentrations, variance parameters of both techniques were comparable. Detected levels of fetal cfDNA in maternal plasma were slightly higher than expected and correlated significantly with gestational age as measured by both methods (ddPCR r = 0.459; qPCR r = 0.438).     

Genomic DNA extraction methods using formalin-fixed paraffin-embedded tissue

As new technologies come within reach for the average cytogenetic laboratory, the study of chromosome structure has become increasingly more sophisticated. Resolution has improved from karyotyping (in which whole chromosomes are discernible) to fluorescence in situ hybridization and comparative genomic hybridization (CGH, with which specific megabase regions are visualized), array-based CGH (aCGH, examining hundreds of base pairs), and next-generation sequencing (providing single base pair resolution). Whole genome next-generation sequencing remains a cost-prohibitive method for many investigators. Meanwhile, the cost of aCGH has been reduced during recent years, even as resolution has increased and protocols have simplified. However, aCGH presents its own set of unique challenges. DNA of sufficient quantity and quality to hybridize to arrays and provide meaningful results is required. This is especially difficult for DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Here, we compare three different methods for acquiring DNA of sufficient length, purity, and “amplifiability” for aCGH and other downstream applications. Phenol–chloroform extraction and column-based commercial kits were compared with adaptive focused acoustics (AFA). Of the three extraction methods, AFA samples showed increased amplicon length and decreased polymerase chain reaction (PCR) failure rate. These findings support AFA as an improvement over previous DNA extraction methods for FFPE tissues.     

Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens

The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material.     

Screening ancient tuberculosis with qPCR: challenges and opportunities

The field of ancient DNA (aDNA) has rapidly accelerated in recent years as a result of new methods in next-generation sequencing, library preparation and targeted enrichment. Such research is restricted, however, by the highly variable DNA preservation within different tissues, especially when isolating ancient pathogens from human remains. Identifying positive candidate samples via quantitative PCR (qPCR) for downstream procedures can reduce reagent costs, increase capture efficiency and maximize the number of sequencing reads of the target. This study uses four qPCR assays designed to target regions within the Mycobacterium tuberculosis complex (MTBC) to examine 133 human skeletal samples from a wide geographical and temporal range, identified by the presence of skeletal lesions typical of chronic disseminated tuberculosis. Given the inherent challenges working with ancient mycobacteria, strict criteria must be used and primer/probe design continually re-evaluated as new data from bacteria become available. Seven samples tested positive for multiple MTBC loci, supporting them as strong candidates for downstream analyses. Using strict and conservative criteria, qPCR remains a fast and effective screening tool when compared with screening by more expensive sequencing and enrichment technologies.     

基于SYBR Green I的双链DNA定量方法

摘要 基于SYBR Green I荧光染料与双链DNA（dsDNA）结合产生荧光的原理,建立一种高精度、高通量的双链DNA 定量方法。将梯度稀释后的基因组DNA及已知浓度的?DNA与等体积的SYBR Green I（4×）充分混合后,利用荧光定量PCR仪采集荧光信号,以ROX（1×）作为校正染料进行定量分析;同时利用紫外分光光度计对样品进行平行测定,比较该方法与紫外分光光度法的检测限与准确度。紫外分光光度法的检测限为2 ng/?l,而SYBR Green I荧光定量法的检测限可达到0.015 ng/?l,并且在0.015~2 ng/?l范围内,SYBR Green I荧光强度与?DNA浓度呈线性关系（R2=0.9999）,比紫外分光光度法灵敏100倍以上,并可准确定量低纯度的DNA样品。此方法具有重复性好、高通量的特点,仅需少量的生物样本即可满足定量要求,为分子生物学研究及临床检验等多个领域提供了一种可靠的dsDNA定量方法。     

Humic substances cause fluorescence inhibition in real-time polymerase chain reaction

Real-time polymerase chain reaction (qPCR) is the cornerstone of DNA analysis, enabling detection and quantification of minute nucleic acid amounts. However, PCR-based analysis is limited, in part, by the presence of inhibitors in the samples. PCR inhibition has been viewed solely as failure to efficiently generate amplicons, that is, amplification inhibition. Humic substances (HS) are well-known inhibitors of PCR amplification. Here we show that HS from environmental samples, specifically humic acid (HA), are very potent detection inhibitors, that is, quench the fluorescence signal of double-stranded DNA (dsDNA) binding dyes. HA quenched the fluorescence of the commonly used qPCR dyes EvaGreen, ResoLight, SYBR Green I, and SYTO 82, generating lowered amplification plots, although amplicon production was unaffected. For EvaGreen, 500 ng of HA quenched nearly all fluorescence, whereas 1000 ng of HA completely inhibited amplification when applying Immolase DNA polymerase with bovine serum albumin (BSA). Fluorescence spectroscopy measurements showed that HA quenching was either static or collisional and indicated that HA bound directly to the dye. Fulvic acid did not act as a qPCR detection inhibitor but inhibited amplification similarly to HA. Hydrolysis probe fluorescence was not quenched by HA. Detection inhibition is an overlooked phenomenon that needs to be considered to allow for development of optimal qPCR assays.     

An improved boiling method for the preparation of bacterial plasmid and phage DNA

A streamlined, reproducible boiling method for preparing plasmid as well as phage replicative form DNA is described. Both quantity and quality of DNA purified are sufficient for restriction enzyme analysis and sequencing. A small loopful of bacteria will provide enough DNA for several experiments, and multiple samples can be processed and prepared for digestion or sequencing in under 20 minutes. DNA preparations are stable for at least 6 months at −20°C.