Method Validation for Extraction of DNA from Human Stool Samples for Downstream Microbiome Analysis.

Background: A formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. A previously optimized stool processing protocol was validated for fitness-for-purpose for downstream microbiome analysis. Materials and Methods: DNA extraction from human stool was validated with various collection tubes, stabilizing solutions and storage conditions in terms of fitness-for-purpose for downstream microbiome analysis, robustness, and sample stability. Acceptance criteria were based on accurate identification of a reference material, homogeneity of extracted samples, and sample stability in a 2-year period. Results: The automated DNA extraction using the chemagic™ Magnetic Separation Module I (MSM I) extracted 8 out of 8 bacteria in the ZymoBIOMICS® Microbial Community Standard. Seven tested stabilizing solutions (OMNIgene®â€¢GUT, RNAlater®, AquaStool™, RNAssist, PerkinElmer SEB lysis buffer, and DNA Genotek's CP-150) were all compatible with the chemagic MSM I and showed no significant difference in microbiome alpha diversity and no significant difference in the overall microbiome composition compared to the baseline snap-frozen stool sample. None of the stabilizing solutions showed intensive polymerase chain reaction (PCR) inhibition in the SPUD assay. However, when we take into account more stringent criteria which include a higher double-stranded DNA yield, higher DNA purity, and absence of PCR inhibition, we recommend the use of OMNIgene•GUT, RNAlater, or AquaStool as alternatives to rapid freezing of samples. The highest sample homogeneity was achieved with RNAlater- and OMNIgene•GUT -stabilized samples. Sample stability after a 2-year storage in -80°C was seen with OMNIgene•GUT -stabilized samples. Conclusions: We validated a combination of a stool processing method with various collection methods, suitable for downstream microbiome applications. Sample collection, storage conditions and DNA extraction methods can influence the microbiome profile results. Laboratories and biobanks should ensure that these conditions are systematically recorded in the scope of accreditation.

Standardization of the preanalytical phase of DNA extraction from fixed tissue for next-generation sequencing analyses.

Next-generation sequencing (NGS) analyses on DNA derived from archived Formalin-Fixed Paraffin-Embedded (FFPE) clinical material can provide a powerful tool in oncology research and clinical diagnostics. Although several studies have established that NGS can be performed using DNA from FFPE tissue, the accuracy and reproducibility of such analyses, as well as their robustness to the biomolecular quality of the samples used, remains a matter of debate. Excellent reviews have recently been published, providing evidence-based best practices for FFPE DNA extraction. Alternative fixatives exist, although their implementation in clinical practice is difficult. In this article, we present (i) a review of fixed tissue DNA preanalytics with a special focus on DNA extraction and fixed tissue sample qualification and (ii) results from comparisons between different methods of DNA extraction from tissue samples that have been fixed or stabilized by different methods, in terms of NGS metrics and different DNA quality metrics.

Preanalytical robustness of blood collection tubes with RNA stabilizers.

Background Efficient blood stabilization is essential to obtaining reliable and comparable RNA analysis data in preclinical operations. PAXgene (Qiagen, Becton Dickinson) and Tempus (Applied Biosystems, Life Technologies) blood collection tubes with RNA stabilizers both avoid preanalytical degradation of mRNA by endogenous nucleases and modifications in specific mRNA concentrations by unintentional up- or down-regulation of gene expression. Methods Sixteen different preanalytical conditions were tested in PAXgene and Tempus blood samples from seven donors: different mixing after collection, different fill volumes and different 24-h transport temperature conditions after collection. RNA was extracted by column-based methods. The quality of the extracted RNA was assessed by spectrophotometric quantification, A260/A280 purity ratio, RNA Integrity Number (Agilent Bioanalyzer), miRNA quantative real time polymerase chain reaction (qRT-PCR) on two target miRNAs (RNU-24 and miR-16), mRNA quality index by qRT-PCR on the 3' and 5' region of the GAPDH gene, and the PBMC preanalytical score, based on the relative expression levels of the IL8 and EDEM3 coding genes. Results When PAXgene RNA and Tempus blood collection tubes were used following the manufacturers' instructions, there was no statistically or technically significant difference in the output RNA quality attributes. However, the integrity of the RNA extracted from Tempus collection tubes was more sensitive to fill volumes and effective inversion, than to storage temperature, while the integrity of RNA extracted from PAXgene collection tubes was more sensitive to effective inversion and storage temperature than to fill volumes. Conclusions Blood collection tubes with different RNA stabilizers present different robustness to common preanalytical variations.

Method Validation for Extraction of Nucleic Acids from Peripheral Whole Blood.

BACKGROUND: This article is the fifth in a series of publications providing formal method validation for biospecimen processing. We report the optimization and validation of methodology to obtain nucleic acids of sufficient quantity and quality from blood. METHODS: DNA was extracted using the Chemagic DNA Blood Kit on an MSM I. Extraction was optimized in terms of blood volume, elution buffer volume, and lysis conditions. The optimal protocol was validated for reproducibility, robustness (delay to buffy coat extraction, blood vs. buffy coat, and use of a magnetic rack), and performance (yield, purity, and concentration). RNA was extracted using a PAXgene Blood miRNA kit with a QiaCube. The protocol was validated for reproducibility, robustness (elution buffer, delay, and temperature before extraction), and performance (yield, purity, integrity, and miRNA content). Two platforms (QiaCube, Biorobot Universal) were further compared. RESULTS: For DNA extraction, a 4 mL blood sample, manual lysis, and 300 µL elution buffer were found to be reproducible (CV <10% for DNA yield and A260 nm/A280 nm ratio) and robust (buffy coat vs. whole blood; immediate processing of buffy coat after lysis vs. storage for 1 week at 2-8°C; and magnetic rack use). There was no difference between automated and manual lysis. RNA extracted with the PAXgene Blood miRNA kit on a QiaCube gave high yields and optimal reproducibility (low CV for RNA yield and integrity) with BR5 elution buffer (vs. water and TE). PAXgene tubes could be stored for up to 2 weeks at 2-8°C. The Biorobot Universal System gave similar mean RNA yields with Qiacube and slightly lower but acceptable purity. CONCLUSIONS: We validated automated isolation of DNA with a Chemagic DNA Blood Kit on a magnetic bead-based MSM I, and of RNA with a PAXgene Blood miRNA kit on a silica membrane-based QiaCube or Biorobot (for low and high throughput, respectively).

Method validation for automated isolation of viable peripheral blood mononuclear cells.

BACKGROUND: This article is part of a series of publications providing formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks. We report the optimization and validation for fitness-for-purpose of automated and manual protocols for isolating peripheral blood mononuclear cells (PBMCs) from whole blood, and compare the two methods. METHODS: The manual method was optimized for whole blood centrifugation speed, gradient type (Ficoll, Leucosep, CPT), and freezing method (Mr Frosty, Controlled Rate Freezing). Various parameters of the automated protocol using a CPT gradient on a Tecan liquid handler were optimized. Optimal protocols were validated in parallel for reproducibility and robustness. Optimization and validation were assessed in terms of cell yield, viability, recovery, white blood cell (WBC) subpopulation distribution, gene expression, and lymphoblastoid cell line (LCL) transformation. RESULTS: An initial centrifugation of whole blood at 2000 g was considered optimal for further processing, allowing isolation of plasma and PBMCs from a single sample. The three gradients gave similar outcomes in terms of cell yield, viability, and WBC subpopulation distribution. Ficoll showed some advantages and was selected for further evaluations. Optimization of the automated protocol script using a CPT gradient gave 61% cell recovery. No significant differences in quality, quantity, and WBC subpopulation distribution were seen between the two freezing methods, and Mr. Frosty was selected. The manual and automated protocols were reproducible in terms of quantity, recovery, viability, WBC subpopulation distribution, gene expression, and LCL transformation. Most (75%-100%) of the 13 robustness parameters were accepted for both methods with an 8 h pre-centrifugation delay versus 38%-85% after 24 h. Differences identified between the automated and manual methods were not considered consequential. CONCLUSIONS: We validated the first fully automated method for isolating viable PBMCs, including RNA analysis and generation of LCLs. We recommend processing within 8 h of blood collection.

Method optimization for fecal sample collection and fecal DNA extraction.

BACKGROUND: This is the third in a series of publications presenting formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks. We report here optimization of a stool processing protocol validated for fitness-for-purpose in terms of downstream DNA-based analyses. METHODS: Stool collection was initially optimized in terms of sample input quantity and supernatant volume using canine stool. Three DNA extraction methods (PerkinElmer MSM I®, Norgen Biotek All-In-One®, MoBio PowerMag®) and six collection container types were evaluated with human stool in terms of DNA quantity and quality, DNA yield, and its reproducibility by spectrophotometry, spectrofluorometry, and quantitative PCR, DNA purity, SPUD assay, and 16S rRNA gene sequence-based taxonomic signatures. RESULTS: The optimal MSM I protocol involves a 0.2 g stool sample and 1000 µL supernatant. The MSM I extraction was superior in terms of DNA quantity and quality when compared to the other two methods tested. Optimal results were obtained with plain Sarstedt tubes (without stabilizer, requiring immediate freezing and storage at -20°C or -80°C) and Genotek tubes (with stabilizer and RT storage) in terms of DNA yields (total, human, bacterial, and double-stranded) according to spectrophotometry and spectrofluorometry, with low yield variability and good DNA purity. No inhibitors were identified at 25 ng/µL. The protocol was reproducible in terms of DNA yield among different stool aliquots. CONCLUSIONS: We validated a stool collection method suitable for downstream DNA metagenomic analysis. DNA extraction with the MSM I method using Genotek tubes was considered optimal, with simple logistics in terms of collection and shipment and offers the possibility of automation. Laboratories and biobanks should ensure protocol conditions are systematically recorded in the scope of accreditation.