Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples

Kresse, Stine H.; Namløs, Heidi M.; Lorenz, Susanne; Berner, Jeanne-Marie; Myklebost, Ola; Bjerkehagen, Bodil; Meza‐Zepeda, Leonardo A.

doi:10.1371/journal.pone.0197456

Cited by 53 publications

(41 citation statements)

References 17 publications

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“…Two tissue sections were stained with hematoxylin and eosin, and the presence of representative tumor material (>60% tumor cells) was verified by a sarcoma pathologist. Genomic DNA was isolated using the Allprep DNA/RNA Kit (Qiagen) and the truXTRAC FFPE DNA Kit (Covaris Inc.) from fresh frozen tumor and formalin-fixed paraffin-embedded (FFPE) tissue (20), respectively.…”

Section: Extraction and Quantification Of Dnamentioning

confidence: 99%

Noninvasive Detection of ctDNA Reveals Intratumor Heterogeneity and Is Associated with Tumor Burden in Gastrointestinal Stromal Tumor

Namløs

Boye

Mishkin³

et al. 2018

Molecular Cancer Therapeutics

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Molecular analysis of circulating tumor DNA (ctDNA) has a large potential for clinical application by capturing tumor-specific aberrations through noninvasive sampling. In gastrointestinal stromal tumor (GIST), analysis of and mutations is important for therapeutic decisions, but the invasiveness of traditional biopsies limits the possibilities for repeated sampling. Using targeted next-generation sequencing, we have analyzed circulating cell-free DNA from 50 GIST patients. Tumor-specific mutations were detected in 16 of 44 plasma samples (36%) from treatment-naïve patients and in three of six (50%) patients treated with tyrosine kinase inhibitors. A significant association between detection of ctDNA and the modified National Institutes of Health risk classification was found. All patients with metastatic disease had detectable ctDNA, and tumor burden was the most important detection determinant. Median tumor size was 13.4 cm for patients with detectable mutation in plasma compared with 4.4 cm in patients without detectable mutation ( = 0.006). ctDNA analysis of a patient with disease progression on imatinib revealed that multiple resistance mutations were synchronously present, and detailed analysis of tumor tissue showed that these were spatially distributed in the primary tumor. Plasma samples taken throughout the course of treatment demonstrated that clonal evolution can be monitored over time. In conclusion, we have shown that detection of GIST-specific mutations in plasma is particularly feasible for patients with high tumor burden. In such cases, we have demonstrated that mutational analysis by use of liquid biopsies can capture the molecular heterogeneity of the whole tumor, and may guide treatment decisions during progression. .

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Section: Extraction and Quantification Of Dnamentioning

confidence: 99%

Noninvasive Detection of ctDNA Reveals Intratumor Heterogeneity and Is Associated with Tumor Burden in Gastrointestinal Stromal Tumor

Namløs

Boye

Mishkin³

et al. 2018

Molecular Cancer Therapeutics

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“…However, no consensus has yet been reached to determine the exact moment in which RNA quality and integrity begin to decrease. Probably this fact is due to the interaction of other pre-analytical factors such as preservation type 12 , preservation method 13 , long term storage 14,15 , RNA extraction methodology 16 , among others.…”

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confidence: 99%

Impact of different stabilization methods on RT-qPCR results using human lung tissue samples

Esteva-Socias

Gómez-Romano

Carrillo-Ávila

et al. 2020

Sci Rep

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Aiming to increase the reproducibility of biomedical research results, biobanks obtain human tissues of the highest quality and carry out different storage methods adapted to the needs of analytical technique to be performed by the biomedical researchers. However, there is much controversy and little data concerning the real impact of different stabilization methods on tissue quality, integrity and functionality of derived biomolecules. The influence of four stabilization methods [RNAlater (RNL), snap freezing (SF), snap freezing using Optimal Cutting Tissue compound (SF-OCT) and formalin-fixed paraffin-embedded (FFPE)] on RNA quality and integrity was evaluated in paired samples of lung tissue. RNA integrity was evaluated through PCR-endpoint assays amplifying six fragments of different length of the HPRT1 gene and RNA Integrity Number (RIN). To evaluate the difference of tissue functionality among the stabilization methods tested, RT-qPCRs were performed focusing on the differential expression of the HPRT1, SNRPD3 and Jun genes. RNA from the samples preserved with the RNL or SF-OCT method showed better integrity compared to SF and FFPE, measured by PCR-endpoint and RT-qPCR assays. However, only statistically significant differences were observed between the RNA from FFPE and other stabilization methods when gene expression of HPRT1, SNRPD3 and Jun housekeeping genes were determined by RT-qPCR. For the three mentioned genes, Cq and RIN values were highly correlated. The present work describes the fragility of SF samples, being critical the moment just before RNA extraction, although further experiments of tissue RNA are needed. Standardization pre-analytic workflow can lead to improved reproducibility between biomedical research studies. The present study demonstrated clear evidences about the impact of the stabilization method on RNA derived from lung human tissue samples.diluted cDNA and 0,6 µl of PCR-grade water. All PCRs were performed in triplicate from one sample taken from each preservation condition in all 20 patients included. PCR reactions were carried out in 96 well plates and using the CFX96 Real-Time PCR Detection System (Bio-Rad) following the steps: 95 °C for 2 minutes, 40 cycles of amplification (denaturation at 95 °C for 5 seconds, annealing for 10 seconds, and extension at 72 °C for 20 seconds) and final melt curve analysis from 65 °C to 95 °C to exclude unspecific PCR products.The reproducibility and dynamic ranges for qPCR assays were determined by constructing a standard curve, beginning with 100 ng of RNA template as initial concentration and then serial diluted 1/5 until five linear concentration points. For Cq values acquisition, RFU threshold value was adjusted to 50 RFUs for each run.Statistics. All data generated from integrity and functional studies of RNA samples were analysed using multiple comparison tests: ANOVA test with Bonferroni correction to detect significant differences between procedures with at least a signification of p value <0.01. The spearman r index between RNA integ...

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“…34 We objectively compared existing lysis protocols with AFA, which is widely used in other NGS domains but has not been explored as a microbiome tool. [20][21][22] AFA technology has characteristics that led us to consider it as a logical choice for processing microbial samples, such as precise scalability across a broad range of energy levels, isothermal lysis conditions and the ability to process especially lysis-resistant bacterial species by transducing large amounts of acoustic energy. Using identical aliquots of a mouse stool homogenate as the substrate, we found that a one-step AFA protocol yields more DNA and uncovers greater microbiome diversity relative to bead beating.…”

Section: Discussionmentioning

confidence: 99%

“…19 More specifically, AFA employs focused bursts of high-frequency ultrasonic acoustic energy (wavelength 1-3 mm) focused into a discrete zone within a sample vessel held at nearisothermal conditions (thus enabling cell wall lysis without heat-induced damage of nucleic acids). Although AFA has found widespread use in other NGS protocols, [20][21][22] our study represents the first evaluation for use in microbiome sequencing. In addition to the side-by-side comparison of AFA with traditional chemical/mechanical protocols (where a single lysis step was used for both methods), we also tested the efficacy of a sequential AFA-based microbiome lysis technique, which aims to account for variation in bacterial cell wall thickness and rigidity in a microbiome sample.…”

Section: Introductionmentioning

confidence: 99%

Hidden components of microbiome diversity revealed by cell wall lysis with Adaptive Focused Acoustics

Wallace

Ahluwalia

Thovarai

et al. 2020

Preprint

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Within the rapidly evolving field of microbiome sequencing, a primary need exists for experimentally capturing microbiota in a manner as close as possible to their in vivo composition. During microbiome profiling, the first step necessarily involves lysis of the cell wall, releasing nucleic acids for next-generation sequencing. Microbial cell wall thicknesses can vary between 5nm to 80nm; while some species are quite easy to lyse, others are particularly resistant to lysis. Despite this, current chemical/mechanical lysis protocols ignore the possibility that species with different cell wall thicknesses are lysed at differential rates. This creates noise in species compositions and possibly skews current microbiome results in ways that are not currently understood. To develop a cell wall thickness-agnostic lysis protocol, we used Adaptive Focused Acoustics (AFA), a tunable acoustic methodology for processing of biological samples.Using identical aliquots of mouse stool homogenate as the lysis substrate, we compared AFA with chemical/mechanical lysis methodology routinely used in microbiome studies and found that AFA-mediated lysis substantially increases both microbial DNA yield as well as alpha and beta diversity. By starting with lower AFA energy levels, sequentially removing aliquots at each step, and subjecting the remainder to progressively stronger AFA treatment, we developed a sequential lysis method that accounts for differences in cell wall thickness. This method revealed even greater levels of diversity than single-timepoint AFA treatment. 16S sequencing results from the above experiments were verified by shotgun metagenome sequencing of a subset of the AFA samples. We found that lysis-induced noise affects not just species compositions, but also functional characterization of shotgun metagenome data. AFA samples also showed a higher detection of eukaryotic and fungal DNA. We suggest that AFA-mediated lysis produces a truer representation of the native microbiota, and that this method deserves consideration as a potential addition to microbiome lysis protocols.

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Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples

Cited by 53 publications

References 17 publications

Noninvasive Detection of ctDNA Reveals Intratumor Heterogeneity and Is Associated with Tumor Burden in Gastrointestinal Stromal Tumor

Noninvasive Detection of ctDNA Reveals Intratumor Heterogeneity and Is Associated with Tumor Burden in Gastrointestinal Stromal Tumor

Impact of different stabilization methods on RT-qPCR results using human lung tissue samples

Hidden components of microbiome diversity revealed by cell wall lysis with Adaptive Focused Acoustics

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