Recovery Of Nucleic Acids Research Articles

Reverse Transcriptase Real Time PCR Detection of Rift Valley Fever Virus RNA in Formalin‐Fixed, Paraffin‐Embedded Tissues

Rift Valley Fever Virus (RVFV) is a mosquito‐borne zoonotic pathogen endemic in parts of Africa and the Arabian Peninsula. RVFV causes acute febrile disease with mass abortion and death in ruminants and occasional encephalitis and death in humans. In the US, RVFV is a BSL‐3Ag Select Agent and a foreign animal disease, requiring special containment laboratories for research work. Research goals include the study of RVFV pathogenesis and the development of novel diagnostic tools and disease prevention methods such as vaccines. Ongoing surveillance plays a critical role in prevention and control of outbreaks and virus spread. Formalin‐fixed, paraffin‐embedded tissues (FFPET) are non‐infectious diagnostic specimens, routinely used for histopathology and immunohistochemistry (IHC), however use of this diagnostic specimen for molecular detection of pathogen RNA is underinvestigated. The objective of this work is to develop and validate a protocol for use of FFPET for the sensitive and specific detection of RVFV RNA.Study samples were fresh‐frozen tissues (FFT) and FFPET (liver, spleen, lung, kidney) collected from sheep and calves experimentally infected with two RVFV strains (Kenya 2006 and Saudi Arabia 2001). Deparaffinization of paraffin whorls (5‐5μm thick), tissue lysis with proteinase K, and reversal of nucleic acid cross linking in heated alkaline buffer was performed in a single tube using commercial buffers (Qiagen). RNA was purified using automated magnetic bead extraction on the Taco mini (GeneReach) for FFPET or the Applied Biosystems reagents on the Kingfisher (FFT). Multiplex Reverse Transcriptase Real Time PCR (RT‐qPCR) for RVFV L, M and S gene segments was used to detect RVFV RNA using Quanta qScript XLT One‐Step RT‐qPCR ToughMix (FFPET) or AgPath RT‐qPCR mix (FFT). IHC for RVFV antigen was done with a mouse monoclonal anti‐RVFV nucleoprotein antibody and avidin‐biotin complex detection.To date, 18 FFPET samples have been tested. For samples that were both IHC and FFT RT‐qPCR positive (n=13), FFPET results were 100% positive. For FFT samples (n=5) that were not tested by RT‐qPCR but were IHC positive, the FFPET RVFV RT‐qPCR results were also positive. Testing of an additional 46 FFPE samples is presently underway.Using this newly developed single tube FFPET technique for nucleic acid recovery, followed by a rapid, low cost magnetic bead nucleic acid extraction allows for highly sensitive detection of RNA specific for RVFV. This novel method could strengthen routine diagnostics, surveillance and molecular epidemiological studies that are currently limited by the strict biosecurity requirements for RVFV samples. Moreover, this methodology could also be expanded for detection of RNA targets for additional high impact pathogens or for transcriptional studies that must relie on archival FFPET.Support or Funding InformationThis work was funded by startup funds provided to Dr. Davis by the Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University and uses archival animal study materials from research funded by grants from the Department of Homeland Security Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD), Grant No. 2010‐ST061‐AG0001, USDA Agricultural Services project 3020‐32000‐005‐00D and the Kansas Bioscience Authority.

Panicum spikelets from the Early Holocene Takarkori rockshelter (SW Libya): Archaeo-molecular and -botanical investigations

This paper deals with the extraction, amplification and sequencing of ancient DNA (aDNA) from spikelets of wild cereals dated at ca. 9000 cal yr BP, representing the most ancient plants with preserved genetic material from the Sahara desert. The sub-fossil records were collected from the archaeological excavation carried out at Takarkori, an archaeological site located in south-western Libya. Morphological and genetic analyses were made on 100 well preserved dried spikelets. Ten DNA extraction protocols were performed to evaluate nucleic acid recovery in terms of DNA yield, purity and amplification success of the chloroplast barcode region matK. The extraction protocol that returned the most suitable DNA to be amplified is the Kistler and Shapiro (2011: J Archaeol Sci 38: 3549-3554) modified protocol. In our study, the results from matK amplification suggested that four specimens are the most appropriate number of spikelets for these analyses. DNA was then used for PCR amplifications of four chloroplast barcode genes: rbcL, matK, trnH-psbA and trnL. A phylogenetic analysis shows the strict relation between the archaeological specimens and modern Panicoideae, supporting the morphological identification. The results indicate that spikelets have a close relation to Panicum laetum Kunth, a wild cereal still collected in tropical Africa.

A melanin-bleaching methodology for molecular and histopathological analysis of formalin-fixed paraffin-embedded tissue

Removal of excessive melanin from heavily pigmented formalin-fixed paraffin-embedded (FFPE) melanoma tissues is essential for histomorphological and molecular diagnostic assessments. Although there have been efforts to address this issue, current methodologies remain complex and time-consuming, and are not suitable for multiple molecular applications. Herein, we have developed a robust and rapid melanin-bleaching methodology for FFPE tissue specimens. Our approach is based on quick bleaching (15 min) at high temperature (80 °C) with 0.5% diluted hydrogen peroxide (H2O2) in Tris-HCl, PBS, or Tris/Tricine/SDS buffer. Immunostaining for Ki-67 and HMB45 was enhanced by bleaching with 0.5% H2O2 in Tris/Tricine/SDS and Tris-HCl, respectively. In addition to histopathological applications, our approach also facilitates recovery of protein and nucleic acid from archival melanin-rich FFPE tissue sections. Protein extracted from bleached FFPE tissues was compatible with western blotting using anti-human GAPDH and AKT antibodies. Our bleaching condition significantly improved RNA quality compared with unbleached tissues without compromising the yield. Notably, the RNA/DNA obtained from bleached tissues was suitable for end point PCR and real-time quantitative RT–PCR. In conclusion, this improved melanin-bleaching method enhances and simplifies immunostaining procedures, and facilitates the use of melanin-rich FFPE tissues for histomorphological and PCR amplification-based molecular assays.

A robust ambient temperature collection and stabilization strategy: Enabling worldwide functional studies of the human microbiome

As reports on possible associations between microbes and the host increase in number, more meaningful interpretations of this information require an ability to compare data sets across studies. This is dependent upon standardization of workflows to ensure comparability both within and between studies. Here we propose the standard use of an alternate collection and stabilization method that would facilitate such comparisons. The DNA Genotek OMNIgene∙Gut Stool Microbiome Kit was compared to the currently accepted community standard of freezing to store human stool samples prior to whole genome sequencing (WGS) for microbiome studies. This stabilization and collection device allows for ambient temperature storage, automation, and ease of shipping/transfer of samples. The device permitted the same data reproducibility as with frozen samples, and yielded higher recovery of nucleic acids. Collection and stabilization of stool microbiome samples with the DNA Genotek collection device, combined with our extraction and WGS, provides a robust, reproducible workflow that enables standardized global collection, storage, and analysis of stool for microbiome studies.

Selective local lysis and sampling of live cells for nucleic acid analysis using a microfluidic probe.

Heterogeneity is inherent to biology, thus it is imperative to realize methods capable of obtaining spatially-resolved genomic and transcriptomic profiles of heterogeneous biological samples. Here, we present a new method for local lysis of live adherent cells for nucleic acid analyses. This method addresses bottlenecks in current approaches, such as dilution of analytes, one-sample-one-test, and incompatibility to adherent cells. We make use of a scanning probe technology - a microfluidic probe - and implement hierarchical hydrodynamic flow confinement (hHFC) to localize multiple biochemicals on a biological substrate in a non-contact, non-destructive manner. hHFC enables rapid recovery of nucleic acids by coupling cell lysis and lysate collection. We locally lysed ~300 cells with chemical systems adapted for DNA or RNA and obtained lysates of ~70 cells/μL for DNA analysis and ~15 cells/μL for mRNA analysis. The lysates were introduced into PCR-based workflows for genomic and transcriptomic analysis. This strategy further enabled selective local lysis of subpopulations in a co-culture of MCF7 and MDA-MB-231 cells, validated by characteristic E-cadherin gene expression in individually extracted cell types. The developed strategy can be applied to study cell-cell, cell-matrix interactions locally, with implications in understanding growth, progression and drug response of a tumor.

DNA and RNA Extraction and Quantitative Real-Time PCR-Based Assays for Biogas Biocenoses in an Interlaboratory Comparison.

Five institutional partners participated in an interlaboratory comparison of nucleic acid extraction, RNA preservation and quantitative Real-Time PCR (qPCR) based assays for biogas biocenoses derived from different grass silage digesting laboratory and pilot scale fermenters. A kit format DNA extraction system based on physical and chemical lysis with excellent extraction efficiency yielded highly reproducible results among the partners and clearly outperformed a traditional CTAB/chloroform/isoamylalcohol based method. Analytical purpose, sample texture, consistency and upstream pretreatment steps determine the modifications that should be applied to achieve maximum efficiency in the trade-off between extract purity and nucleic acid recovery rate. RNA extraction was much more variable, and the destination of the extract determines the method to be used. RNA stabilization with quaternary ammonium salts was an as satisfactory approach as flash freezing in liquid N2. Due to co-eluted impurities, spectrophotometry proved to be of limited value for nucleic acid qualification and quantification in extracts obtained with the kit, and picoGreen® based quantification was more trustworthy. Absorbance at 230 nm can be extremely high in the presence of certain chaotropic guanidine salts, but guanidinium isothiocyanate does not affect (q)PCR. Absolute quantification by qPCR requires application of a reliable internal standard for which correct PCR efficiency and Y-intercept values are important and must be reported.

Protocol for Microfluidic System to Automate the Preparation and Fractionation of the Nucleic Acids in the Cytoplasm Versus Nuclei of Single Cells

This protocol describes the extraction, fractionation, and recovery of cytoplasmic nucleic acids (e.g., cytoplasmic RNA) versus nucleic acids in the cell nucleus (including genomic DNA, gDNA) from single cells with a microfluidic system. The method enables independent, sequence-specific analyses of these critical markers (Kuriyama et al., 2015). The system uses a microfluidic chip with a simple geometry and four end-channel electrodes, and completes the entire process in less than 5 min, including lysis, purification, fractionation, and delivery to two output reservoirs: One for the nucleus (including gDNA and nuclear RNA) and one for cytoplasmic RNA. Each reservoir then contains high quality and purity aliquots with no measurable cross-contamination of cytoplasmic RNA versus nucleic acids in nucleus. As described here, our protocol focuses on the analysis of cytoplasmic RNA versus gDNA from the nucleus. We have tested this protocol with mouse and human cells but not with walled cells such as plant cells.

BioDry: An Inexpensive, Low-Power Method to Preserve Aquatic Microbial Biomass at Room Temperature.

This report describes BioDry (patent pending), a method for reliably preserving the biomolecules associated with aquatic microbial biomass samples, without the need of hazardous materials (e.g. liquid nitrogen, preservatives, etc.), freezing, or bulky storage/sampling equipment. Gel electrophoresis analysis of nucleic acid extracts from samples treated in the lab with the BioDry method indicated that molecular integrity was protected in samples stored at room temperature for up to 30 days. Analysis of 16S/18S rRNA genes for presence/absence and relative abundance of microorganisms using both 454-pyrosequencing and TRFLP profiling revealed statistically indistinguishable communities from control samples that were frozen in liquid nitrogen immediately after collection. Seawater and river water biomass samples collected with a portable BioDry “field unit", constructed from off-the-shelf materials and a battery-operated pumping system, also displayed high levels of community rRNA preservation, despite a slight decrease in nucleic acid recovery over the course of storage for 30 days. Functional mRNA and protein pools from the field samples were also effectively conserved with BioDry, as assessed by respective RT-PCR amplification and western blot of ribulose-1-5-bisphosphate carboxylase/oxygenase. Collectively, these results demonstrate that BioDry can adequately preserve a suite of biomolecules from aquatic biomass at ambient temperatures for up to a month, giving it great potential for high resolution sampling in remote locations or on autonomous platforms where space and power are limited.

Laser Microdissection

Laser microdissection (LM) offers a relatively rapid and precise method of isolating and removing specified cells from complex tissues for subsequent analysis of their RNA, DNA, protein or metabolite content, thereby allowing assessment of the role of different cell types in the normal physiological or disease processes being studied. In this unit, protocols for the preparation of mammalian frozen tissues, fixed tissues, and cytologic specimens for LM, including tissue freezing, tissue processing and paraffin embedding, histologic sectioning, cell processing, hematoxylin and eosin staining, immunohistochemistry, and image-guided cell targeting are presented. Also provided are recipes for generating lysis buffers for the recovery of nucleic acids and proteins. The Commentary section addresses the types of specimens that can be utilized for LM and approaches to staining of specimens for cell visualization. Emphasis is placed on the preparation of tissue or cytologic specimens as this is critical to effective LM.

Commercially available kits for manual and automatic extraction of nucleic acids from formalin-fixed, paraffin-embedded (FFPE) tissues.

Formalin-fixed, paraffin-embedded (FFPE) tissues represent an invaluable source for diagnostic purposes when fresh clinical material is unavailable, and also for molecular and epidemiological studies. The recovery of nucleic acids from FFPE tissues is particularly challenging, and several in-house methods have been developed for this purpose over the last three decades. Recently, several commercial kits specifically developed for DNA and/or RNA extraction from FFPE tissues have been introduced to the market, but their inventory is not available in peer-reviewed literature. This article provides the first comprehensive inventory of commercial FFPE DNA/RNA extraction kits currently available on the market and describes their basic characteristics and features. A total of 69 commercial kits from 43 companies were identified. Thirty-five kits were developed specifically for DNA extraction, 22 for RNA extraction, and 12 for both DNA and RNA extraction. Only two commercial kits allow full automation of the entire nucleic acid extraction procedure. The tissue deparaffinization step is omitted in many protocols by melting paraffin directly in a tissue lysis buffer. Purification of the released nucleic acids is mainly based on silica or resin adsorption technology. A formalin reverse cross-linking step to increase the quality of extracted DNA and RNA is an intrinsic part of over half of the kits identified. It is hope that this comprehensive list of available commercial kits for extracting nucleic acids from FFPE will encourage researchers to strongly consider using them in diagnostic and research settings instead of old-fashioned, crude, and probably less effective in-house methods.

Abstract 4738: Maximize recovery of quality nucleic acid from formalin-fixed paraffin-embedded tissue samples using a novel, flexible purification technology

Abstract Formalin-fixed paraffin-embedded (FFPE) tissue samples are a valuable source of genetic information for gene expression and clinical research. Extraction of nucleic acid (NA) from FFPE tissues is a challenge because the fixation process results in cross-linking between proteins and nucleic acid, as well as between different strands of DNA or RNA molecules. Recent advances in sample preparation enables access to the valuable information contained within these difficult samples. We use a novel purification technology that does not rely on use of any harsh organic solvents and concentrates the NA for low volume elution on a specially designed spin column, thus maximizing concentration. Using this technology, we examined several aspects of the purification protocol for flexibility and adaptability: effect of de-crosslinking time on the quality and length of nucleic acids released from diverse FFPE tissues, effect of overnight storage between lysis and extraction steps, and whether the technology could be adapted to extract both DNA and RNA from the same FFPE tissue sample. Yield and quality of isolated nucleic acids were examined by absorbance, fluorescent binding dye, and real-time qPCR and RT-qPCR. We demonstrate that amplification-based quantitation gave the best indication of the quality of NA recovered. We found that a short decrosslinking time gave significant yields, but only with longer decrosslinking, times were amplifiable yields maximized for many of the tissue types tested. Preprocessing conditions were determined that allow release of both DNA and RNA for subsequent capture and elution in separate spin columns, enabling recovery of high quality DNA and RNA from the same sample in separate tubes. In this study, we demonstrate that de-crosslinking conditions can affect the recovery of amplifiable nucleic acid from FFPE samples and that RNA and DNA can be recovered from the same FFPE sample using an optimized protocol. Citation Format: Doug Wieczorek, Brad Hook, Eric Vincent, Trista Schagat. Maximize recovery of quality nucleic acid from formalin-fixed paraffin-embedded tissue samples using a novel, flexible purification technology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4738. doi:10.1158/1538-7445.AM2015-4738

Recovery of viral RNA and infectious foot-and-mouth disease virus from positive lateral-flow devices.

Foot-and-mouth disease Virus (FMDV) is an economically important, highly contagious picornavirus that affects both wild and domesticated cloven hooved animals. In developing countries, the effective laboratory diagnosis of foot-and-mouth disease (FMD) is often hindered by inadequate sample preservation due to difficulties in the transportation and storage of clinical material. These factors can compromise the ability to detect and characterise FMD virus in countries where the disease is endemic. Furthermore, the high cost of sending infectious virus material and the biosecurity risk it presents emphasises the need for a thermo-stable, non-infectious mode of transporting diagnostic samples. This paper investigates the potential of using FMDV lateral-flow devices (LFDs) for dry transportation of clinical samples for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by electroporation. FMDV positive samples (epithelial suspensions and cell culture isolates) representing four FMDV serotypes were applied to antigen LFDs: after which it was possible to recover viral RNA that could be detected using real-time RT-PCR. Using this nucleic acid, it was also possible to recover VP1 sequences and also successfully utilise protocols for amplification of complete FMD virus genomes. It was not possible to recover infectious FMDV directly from the LFDs, however following electroporation into BHK-21 cells and subsequent cell passage, infectious virus could be recovered. Therefore, these results support the use of the antigen LFD for the dry, non-hazardous transportation of samples from FMD endemic countries to international reference laboratories.

Sensitive, Efficient Quantitation of 13C-Enriched Nucleic Acids via Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry for Applications in Stable Isotope Probing.

Stable isotope probing (SIP) of nucleic acids is a powerful tool for studying the functional traits of microbial populations within complex communities, but SIP involves a number of technical challenges. Many of the difficulties in DNA-SIP and RNA-SIP experiments can be effectively overcome with an efficient, sensitive method for quantitating the isotopic enrichment of nucleic acids. Here, we present a sensitive method for quantitating (13)C enrichment of nucleic acids, requiring a few nanograms of sample, and we demonstrate its utility in typical DNA-SIP and RNA-SIP experiments. All five nucleobases (adenine, guanine, cytosine, thymine, and uracil) were separated and detected by using ultrahigh-performance liquid chromatography-tandem mass spectrometry. We detected all isotopic species in samples with as low as 1.5 atom% (13)C above natural abundance, using 1-ng loadings. Quantitation was used to characterize the isotopic enrichment kinetics of cellulose- and lignin-based microcosm experiments and to optimize the recovery of enriched nucleic acids. Application of our method will minimize the quantity of expensive isotopically labeled substrates required and reduce the risk of failed experiments due to insufficient recovery of labeled nucleic acids for sequencing library preparation.

Improved yield of high molecular weight DNA coincides with increased microbial diversity access from iron oxide cemented sub-surface clay environments.

Despite over three decades of progress, extraction of high molecular weight (HMW) DNA from high clay soils or iron oxide cemented clay has remained challenging. HMW DNA is desirable for next generation sequencing as it yields the most comprehensive coverage. Several DNA extraction procedures were compared from samples that exhibit strong nucleic acid adsorption. pH manipulation or use of alternative ion solutions offered no improvement in nucleic acid recovery. Lysis by liquid N2 grinding in concentrated guanidine followed by concentrated sodium phosphate extraction supported HMW DNA recovery from clays high in iron oxides. DNA recovered using 1 M sodium phosphate buffer (PB) as a competitive desorptive wash was 15.22±2.33 µg DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25 µg DNA/g clay with the Powerlyzer system (MoBio). Increasing PB concentration in the lysis reagent coincided with increasing DNA fragment length during initial extraction. Rarefaction plots of 16S rRNA (V1–V3 region) pyrosequencing from A-horizon and clay soils showed an ∼80% and ∼400% larger accessed diversity compared to the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more operational taxonomic units (OTU) recovered.

Absolute quantification of viral DNA: the quest for perfection.

In spite of the impressive technical refinement of the PCR technology, new-generation real-time PCR assays still suffer from two major limitations: the impossibility to control both for PCR artifacts (with the important caveat of false-negative results) and for the efficiency of nucleic acid recovery during the preliminary extraction phase of DNA from the biological sample. The calibrator technology developed at the Unit of Human Virology overcomes both of these limitations, leading to a substantially higher degree of accuracy and reproducibility in the quantification, which is especially useful for the measurement of pathogen loads in sequential samples and for the reliable detection of low-copy pathogens.

Perspectives in isolation of microRNA from thyroid fine-needle aspiration: reply to the letter "Nucleic acid recovery from thyroid fine-needle cytology slides"

Perspectives in isolation of microRNA from thyroid fine-needle aspiration: reply to the letter “Nucleic acid recovery from thyroid fine-needle cytology slides”

Nucleic acid recovery from thyroid fine-needle cytology slides

M uch interest has been focused in nucleic acid isolation from tissue samples stored for long periods and their utilization on molecular biomarkers research. However, sample fixation induces chemical modifications in macromolecules (1) that result in a laborious and difficult protocol to extract nucleic acid for molecular analysis. In this context, the article recently published by Kizys and cols. (2) contributed to show efficient methods of nucleic acid recovery from archived formalin-fixed/ paraffin-embedded (FFPE) and fine-needle aspiration (FNA) samples of thyroid tissue. Despite the efforts, the issue that remains a challenge in the most effective protocols is to obtain better yield and quality of DNA/RNA due to the damaging effect of the fixation process, particularly for RNA analysis. In contrast with the difficulty in obtaining conserved messenger RNA from archived samples, the fraction of microRNA (miRNA) is less affected by fixation and storage time (3,4). Mature miRNA is a single-stranded noncoding small RNA of ~19-22 nucleotide length that regulates gene expression at the post-transcriptional level (5). These molecules pair to 3’UTRs of target mRNA and thereby cause their silencing or degradation. The miRNA might be a promising prognostic and diagnostic biomarker for thyroid cancer (6). In this context, we have performed, in archived FNA-stained slides, the analysis of BRAFT1799A mutation (7) along with miRNAs expression, miR-146b (8) and let7-f (9), which were modulated in papillary thyroid carcinoma (PTC) (10,11). The acid guanidinium thiocyanate-phenol-chloroform method (12) using TRIzolTM enabled the RNA/DNA co-isolation from the same archived FNA, despite the reduced number of cells in the cytological slides. From the extracted RNA, we performed real-time PCR to amplify miRNA, and observed high expression of miR-146b in PTC compared with goiter (normalized expression miR-146b/RNU6B: PTC 21.55 ± 5.83* vs. goiter 0.18 ± 0.06, * P < 0.05), and no change in let7-f expression (normalized expression let7-f /RNU6B: PTC 0.30 ± 0.13 vs. goiter 0.07 ± 0.02, ns). In addition, DNA obtained was suitable for BRAF mutation analysis by RFLP-PCR (Restriction Fragment Length Polymorphism -PCR) in all FNA slides tested. Using this approach, we observed that it is possible to access both nucleic acids, RNA and DNA, from the same archived thyroid FNA-stained slide. Furthermore, DNA was adequate to perform mutation analysis, and the quality of total RNA and its amount was satisfactory to investigate several miRNAs from the same sample. In conclusion, this procedure could be useful in detecting molecular biomarkers and drawing correlations with cytological analysis, which might improve the accuracy of FNA as a diagnostic tool of thyroid nodule. 1 Department of Cell and Developmental Biology, Institute of Biomedical Sciences, Universidade de Sao Paulo (USP), Sao Paulo, SP, Brazil

A Magnetic Bead-Based Method for Concentrating DNA from Human Urine for Downstream Detection

Due to the presence of PCR inhibitors, PCR cannot be used directly on most clinical samples, including human urine, without pre-treatment. A magnetic bead-based strategy is one potential method to collect biomarkers from urine samples and separate the biomarkers from PCR inhibitors. In this report, a 1 mL urine sample was mixed within the bulb of a transfer pipette containing lyophilized nucleic acid-silica adsorption buffer and silica-coated magnetic beads. After mixing, the sample was transferred from the pipette bulb to a small diameter tube, and captured biomarkers were concentrated using magnetic entrainment of beads through pre-arrayed wash solutions separated by small air gaps. Feasibility was tested using synthetic segments of the 140 bp tuberculosis IS6110 DNA sequence spiked into pooled human urine samples. DNA recovery was evaluated by qPCR. Despite the presence of spiked DNA, no DNA was detectable in unextracted urine samples, presumably due to the presence of PCR inhibitors. However, following extraction with the magnetic bead-based method, we found that ∼50% of spiked TB DNA was recovered from human urine containing roughly 5×103 to 5×108 copies of IS6110 DNA. In addition, the DNA was concentrated approximately ten-fold into water. The final concentration of DNA in the eluate was 5×106, 14×106, and 8×106 copies/µL for 1, 3, and 5 mL urine samples, respectively. Lyophilized and freshly prepared reagents within the transfer pipette produced similar results, suggesting that long-term storage without refrigeration is possible. DNA recovery increased with the length of the spiked DNA segments from 10±0.9% for a 75 bp DNA sequence to 42±4% for a 100 bp segment and 58±9% for a 140 bp segment. The estimated LOD was 77 copies of DNA/µL of urine. The strategy presented here provides a simple means to achieve high nucleic acid recovery from easily obtained urine samples, which does not contain inhibitors of PCR.

Universal extraction method for gastrointestinal pathogens

A universal stool extraction method for recovery of nucleic acids (NAs) from gastrointestinal pathogens was developed to support rapid diagnostics for the London 2012 Olympics. The method involved mechanical disruption (bead beating) of the stools, followed by automated extraction and detection using real-time PCR. This method had been used extensively in the Second Infectious Intestinal Disease Study (IID2) for the isolation of NA from bacteria and parasites (and was effective for the robust recovery of Cryptosporidium spp.) but had not been used for enteric viruses. To ensure this method was universally suitable, panels of samples known to contain target bacteria, viruses or parasites were processed in triplicate using the pre-treatment method routinely used for each target and the new extraction method (bead beating). The extracts were tested using real-time PCR and the cycle threshold values were compared. The results from this study showed that bead beating improved yields for the bacterial and parasitic targets and was suitable for the viral targets. The implementation of this universal method should confer cost- and time-saving benefits and streamline the processes required for the characterization of an array of pathogens from faecal samples.

Perspectives in isolation of microRNA from thyroid fine-needle aspiration: reply to the letter "Nucleic acid recovery from thyroid fine-needle cytology slides"

E fforts have been made to improve the isolation of nucleic acids from long-term preserved samples (1,2). This is a challenge when dealing with formalin-fixed/ paraffin-embedded (FFPE) tissues and fine needle aspiration (FNA) samples from stained slides. In our recent study (3) using in-house protocols, we optimized the methods used for nucleic acid extraction, including co-extraction of RNA/DNA from thyroid FNA in stained slides samples and FFPE thyroid tissues by means of modifications of the standard TRIzol® reagent (Life Technologies, Carlsbad, CA, USA) protocol, making genetic and epigenetic studies possible using the same sample. In addition, the recovery of nucleic acid from the same thyroid FNA stained slides that had been previously analyzed by a cytopathologist enables an integrated diagnosis, as well as repeated FNAs, reducing the costs with cytopathology and molecular procedures. Several studies have been published regarding the potential of microRNA (miRNA) as a molecular biomarker of thyroid cancer (5). miRNA are smaller (~19-22 bp) and more stable than messenger RNA (mRNA), so its detection and recovery are equally feasible, as objectively demonstrated by Saito and cols. In this context, we are honored to know that our article has stimulated Professor Kimura’s team to show their results in miRNA extraction from thyroid FNA stained slides. They were successful with the isolation of RNA and analysis of several miRNAs. Besides, our group and Kimura’s team were able to co-extract DNA/RNA with further BRAF analysis by means of PCR-Sequencing (3) and PCR-RFLP analysis (Saito and cols.). In conclusion, miRNA isolation from thyroid FNA stained slides is another promising tool when investigating thyroid nodules, especially those with uncertain cytopathology results.