Co-extraction Method Research Articles

Development of a multiplex reverse transcription qPCR assay for identification of saliva, blood, and semen

ABSTRACT The identification of body fluids can provide important information for case processing in forensic investigations. Accurate identification through messenger RNA (mRNA) is effective, especially in cases of sexual assault, where body fluids from one or more contributors are mixed. In this study, a saliva, blood, and semen simultaneous identification reverse transcription-quantitative polymerase chain reaction (SBS RT-qPCR) assay using mRNA from a DNA/RNA co-extraction method was developed. The body fluid-specific mRNA markers histatin 3 (HTN3), haemoglobin subunit beta (HBB), and protamine 1 (PRM1) were selected for identification of saliva, blood, and sperm cells, respectively. The assay accurately identified saliva, blood, and semen without cross-reaction with 10 body fluids. The limits of detection (LoD) were 1/102-, 1/105-, and 1/103-fold dilutions of total RNA in saliva, blood, and semen, respectively. Furthermore, the assay accurately identified trace amounts of body fluids in mixed samples at a 58:1:1 ratio. Finally, it was possible to analyse the origin of body fluids by obtaining an STR profile using the DNA/RNA co-extraction method. The SBS RT-qPCR assay was able to simultaneously identify each body fluid with efficiency in sample consumption. Therefore, it is advisable to use the DNA/RNA co-extraction method to identify mixed body fluids in sexual assault cases.

A Knowledge-Integrate Cross-Domain Data Generation Method for Aspect and Opinion Co-Extraction

A Knowledge-Integrate Cross-Domain Data Generation Method for Aspect and Opinion Co-Extraction

A Fast and Easy Method to Co-extract DNA and RNA from an Environmental Microbial Sample.

We herein propose a fast and easy DNA and RNA co-extraction method for environmental microbial samples. It combines bead beating and phenol-chloroform phase separation followed by the separation and purification of DNA and RNA using the Qiagen AllPrep DNA/RNA mini kit. With a handling time of ~3 h, our method simultaneously extracted high-quality DNA (peak size >10-15‍ ‍kb) and RNA (RNA integrity number >6) from lake bacterioplankton filtered samples. The method is also applicable to low-biomass samples (expected DNA or RNA yield <50‍ ‍ng) and eukaryotic microbial samples, providing an easy option for more versatile eco-genomic applications.

Comprehensive recovery of NCM cathode materials for spent lithium-ion batteries by microfluidic device

With the gradual expansion of the market scale of lithium-ion batteries, the importance of comprehensive recycling of lithium-ion battery cathode materials has become increasingly prominent. In this work, a comprehensive recovery of leachate from spent NCM cathode materials was established with the microextraction equipment. In the process of extraction, we established an economical and efficient co-extraction method of Mn-Co-Ni to separate with Li ions in the leachate. After investigating the extraction effects of single extractant of D2EHPA and Cyanex272, the co-extraction pattern of the mixed extractant was summarized by exploring the correlation of extraction efficiency and selectivity with equilibrium pH, extractant concentration ratio and two-phase ratio. The mechanism of saponification and extraction with the mixed extractant was explained based on the IR spectral results. After separately exploring the stripping process and the deep recovery process of Ni, the metal ion of lithium was recycled via precipitation. The regenerated lithium carbonate reached 99.2% purity with uniform particle size distribution. Leveraging the microextraction equipment with greater mass transfer coefficient, a comprehensive extractive recovery process of Mn-Co-Ni was established and realized with the recovery efficiency of Mn, Co, Ni and Li of 99.9%, 98.6%, 99.5% and 90%, respectively. The regenerated metal solution of Mn-Co-Ni can be further used to prepare the precursors of regenerated cathode materials.

Development of a combined differential DNA/RNA co-extraction protocol and its application in forensic casework

In forensic casework, a broad range of extraction techniques is available, and the type of case will decide on the extraction method. Generally, in sexual assault cases, either the presence of semen is questioned (foremost in victim samplings), or the presence of body fluids such as vaginal mucosa or saliva (mostly in samples taken from the suspect). To analyze the semen component, differential extraction (DE) procedures are helpful as spermatozoa DNA gets specifically extracted in a distinct fraction. To assess which body fluid is present, messenger RNA (mRNA) profiling has emerged as one the best methods especially for body fluids encountered in forensic sexual assault cases. mRNA profiling requires the use of DNA/RNA co-extraction methods. In common sexual assault cases, mRNA typing is requested whilst semen may also be present, possibly in surplus amounts. This drove the development of a combined DNA/RNA/DE extraction method that yields DNA and RNA of both the sperm fraction as well as the non-sperm fraction. For the validation of the developed method, its performance was successfully assessed against conventional extraction methods, a range of relevant body fluids and stains up to 1 month old. The developed protocol was implemented for use in casework and is now part of the regular casework workflow in our laboratory.

Discovery of Sex-Determining Genes in Portunus trituberculatus: A Comparison of Male and Female Transcriptomes During Early Developmental Stages

Portunus trituberculatus is one of the main mariculture crabs of high economic value. To identify genes involved in sex determination, we first performed sex-specific transcriptome sequencing at six larval development stages using a DNA/RNA co-extraction method. A total of 907,952,938 and 828,774,880 reads were obtained from female and male crabs, respectively. 2,379 differentially expressed genes (DEGs) were found between females and males, and were mainly enriched in DNA replication, folate biosynthesis, and retinol metabolism pathways. Furthermore, transcription patterns of genes in the sex-determining region (SD) were analyzed based on the transcriptome data, and one Dmrt gene (PtDMY) was found to be exclusively expressed in males during early developmental stages. Notably, some known sex-related genes, including IAG, Dmrt11E, DmrtB1, and DmrtC2 were significantly down-regulated after knocking down PtDMY. Our results suggested that PtDMY is involved in sex determination and may be one of the key upstream regulators of the sex determination pathway. In addition, the massive volume of transcriptome data obtained in this study provided an important basis for the systematic study of sex determination mechanisms in P. trituberculatus.

Two hours method for RNA and DNA co-extraction from blood of coronary artery disease patients: Fast, simple and economical technique.

Objectives:Extraction of DNA and RNA is the first step in genomics and transcriptomics studies. Phenol-chloroform method for DNA extraction has been the widely used method. However, this method is relatively expensive and time-consuming. The objective of the present study was to validate a cost and time-effective protocol that will reduce the burden of molecular biology-based research and make a difference in laboratories with limited resources.Methods:A comparative study was conducted at Syed Qamer Alam Research Laboratory, Shifa College of Medicine; from February, 2021 to August, 2021. TRIzol™ method was used to extract RNA from blood samples of coronary artery disease patients and remnant was used to extract DNA. The quantity, purity and integrity of the extracted DNA by both methods (TRIzol and phenol-chloroform) was examined. PCR product amplification was performed with thrombomodulin (THBD) gene to validate the characteristic of the extracted DNA and its efficiency for downstream experiments.Results:The DNA yield in the TRIzol™ method was three-fold higher than phenol chloroform method. Both methods showed intact genomic DNA on the agarose gel, and extracted DNA was efficient for PCR amplification.Conclusion:The TRIzol™ method for RNA and DNA co-extraction is fast, simple and economical technique. So, it can be adopted for routine molecular biology analyses in limited resources setup.

Evaluating the performance of five up-to-date DNA/RNA co-extraction methods for forensic application

The importance of RNA evidence is growing with new developments in RNA profiling methods and purposes. As forensic samples often can be of small quantity, extraction methods with high yields of both DNA and RNA are desirable. In order to identify the optimal DNA/RNA co-extraction workflow for forensic samples, we evaluated the performance of three frequently-used methods, two new approaches for DNA/RNA co-extraction and a manual phenol/chloroform RNA-only extraction method on blood and saliva samples. Based on a comprehensive analysis of the RNA and DNA quantities, as well as the STR genotyping and mRNA profiling results, we conclude that the two frequently-used co-extraction methods, combining commercially available DNA and RNA extraction kits, achieved the best performance. However, not any combination of commercially available DNA and RNA extraction kits works well and extensive optimization is necessary, as seen in the poor results of the two new approaches.

Sex-Specific Transcriptome Sequencing of Zoea I Larvae and Identification of Sex-Linked Genes Using Bulked Segregant Analysis in Pacific White Shrimp Litopenaeus vannamei.

In order to learn the sex determination and differentiation process in shrimp, we performed sex-specific transcriptome sequencing of the Zoea I larvae in Litopenaeus vannamei (L. vannamei) using a DNA/RNA co-extraction method. The sex-specific genes and the sex-linked SNPs were identified. De novo assembly of all the clean reads generated 41,270 unigenes with an average length of 1026bp and an N50 of 1922bp. A total of 60 differentially expressed genes (DEGs) between females and males at Zoea I stage were obtained, in which 41 unigenes had annotations. DEGs that might be related to sex development in L. vannamei were analyzed. Besides, paralogous genes of some known sex-related genes in model organisms such as sex-lethal (Sxl) showed no sex-biased difference, which indicated they may not play roles in sex development at Zoea I stage. Candidate sex-linked SNPs were validated in six populations of L. vannamei, and SNPs in Unigene0020898 and Unigene0020336 were fully linked to genders, suggesting the two genes located in the sex-determining region of L. vannamei. Our report provided the sex-specific gene expression in early developmental stage of L. vannamei, and found some sex-biased genes that may participate in the sex development process. We also detected two sex-linked genes which may be located in the sex determination region of L. vannamei through bulked segregant analysis, and the SNPs on these genes also provided a method for genetic sex identification at transcriptional level. Our findings will facilitate further researches on molecular mechanism of sex determination and differentiation in shrimps.

One-step coextraction method for flavouring soybean oil with the dried stipe of Lentinus edodes (Berk.) sing by supercritical CO2 fluid extraction

Dried Lentinus edodes (Berk.) sing stipe (LES) is mostly discarded owing to its high fibre content and bad palatability, causing significant waste of resources. The aim of this study is to sustainably use LES for the production of flavour soybean oil using the supercritical CO2 fluid extraction (SFE) method. The quality properties of the obtained soybean oil flavoured by LES (OFLS) were evaluated in this study. Profiles obtained with an electronic nose and gas chromatography results obtained by quadrupole time-of-flight mass spectrometry and with a mass-selective detector indicated that mushroom-like aroma compounds were mainly concentrated into the OFLSs from separation kettle A (9 MPa). Interestingly, off-flavour compounds from soybean oil, such as o-xylene and trans-2-nonenal, were mostly distilled into separation kettle B (4 MPa). More importantly, the developed SFE method effectively improved the colour quality of the OFLSs. Additionally, the decrease of the viscosity of the OFLSs indicated that the thermal stability of the edible oil could be improved. In summary, the OFLSs prepared by the green and sustainable SFE method significantly improved the quality of the edible oil.

Evaluation of a co-extraction kit for mRNA, miRNA and DNA methylation-based body fluid identification.

Recently, messenger RNA (mRNA), micro RNA (miRNA), and DNA methylation (DNAm) have been reported as novel markers for body fluid identification (BFID). Comprehensive analysis of these markers should be a flexible and reliable BFID method for various types of forensic samples. However, independent extraction of all targets can be difficult depending on the usable amounts of samples. In this study, the applicability of a co-extraction kit for these molecules, the AllPrep DNA/RNA/miRNA Universal Kit (APU), was evaluated by comparing RNA and DNA extracted from blood and saliva stains by the APU with those extracted by standard kits for each molecule and by previously reported methods for mRNA/DNA or miRNA/DNA co-extraction. Electrophoresis using the Bioanalyzer platform and real-time PCR analysis revealed that the APU performed almost equivalently to each standard kit in the quality of RNA or DNA extracted and extraction efficiency of mRNAs, miRNAs, and DNA. Moreover, the APU outperformed the co-extraction methods, especially in RNA integrity and miRNA extraction efficiency. In addition, pyrosequencing revealed that the methylation ratios of DNA extracted by the APU were not different from those extracted by standard DNA extraction kits. Overall, the APU is applicable to comprehensive analysis of mRNA/miRNA/DNAm markers for BFID analysis. Because the DNA eluate can also be used for DNA typing, the APU may be among the best choices for forensic examination of body fluid samples in terms of its flexibility and reliability in BFID and efficiency in sample consumption.

Evaluation of two DNA/RNA co-extraction methods for body fluid identification in forensics

Body fluid identification has become a field of interest in forensic casework as it can add value to particular investigative scenarios. Identifying the source of the biological material is not always an upfront task using conventional methods; therefore, profiling of specific mRNA markers can provide the answer. The implementation of RNA based analyses in forensic casework must focus on the quality and sensitivity of methods, starting with nucleic acid extraction, and without loss of DNA for STR profiling. In this work, two methods for DNA and RNA co-extraction were tested and compared: a commercial kit that uses a spin, mini column methodology, and a quick, simple nucleic acid isopropanol precipitation based protocol. Both methods simultaneously extract DNA and RNA, crucial for forensic casework and were tested in semen samples. Nucleic acid quantifications as well as purity assessment ratios (OD260/OD230 and OD260/OD280) were obtained by both methods to infer on the use of extracts in downstream applications such as PCR. The performance of the two tested protocols was further evaluated by analyzing two semen mRNA specific markers, PRM1 and SEMG1. When compared to the commercially developed kit, results suggest that the literature adapted protocol allowed more carryover of contaminants absorbing at 230 nm and 280 nm as the purity ratios were below the accepted standard ranges. Negative results for mRNA profiling supported the QC results obtained by spectrophotometry. On the hand, PRM1 and SEGM1 were positive in RNA samples extracted with the commercial kit.

Abstract 1702: Comprehensive genomic analysis of predictive biomarkers for checkpoint inhibitor therapy from a limited amount of FFPE tissue and cfDNA

Abstract PD-L1 expression and MSI status are useful predictive biomarkers for checkpoint inhibitors (CIs) in some tumor types. However, studies have demonstrated that the identification of all potential patients who may benefit from CIs will require the analysis of both tumor cell and tumor microenvironment (TME) biomarkers. For example, recent data suggests that tumor mutation burden (TMB) and tumor mutation signatures as well as inflammatory signatures and T cell clonality in the tumor microenvironment may serve as independent predictive biomarkers of CI response. The need for evaluation of multiple biomarkers using IHC, PCR and/or NGS using limited amounts of tissue poses a challenge in the clinical setting. Here we conduct a study to evaluate whether a panel of tumor and TME biomarkers can be accurately analyzed using low DNA and RNA input from FFPE specimens or using plasma-derived cfDNA. Multiple methods for assessing TMB, MSI status and mutation signatures were compared. The impact of sample quality on biomarker analysis was also evaluated. A set of late stage CRC and matched plasma samples were used in this study. DNA and RNA were co-extracted from FFPE sections using RecoverAll™ kit. cfDNA was extracted from plasma using the MagMAX™ Kit. DNA and RNA yields from FFPE ranged from 855-3875ng and 477-5200ng, respectively. cfDNA yields ranged from 8-38ng. Based on the KAPA DNA QC results, the FFPE samples were determined to be high quality. TMB and mutation signature analysis of FFPE samples were performed using Oncomine Tumor Mutation Load (TML) Assay, the Oncomine Comprehensive Assay v3 (OCAv3) and WES. MSI status in FFPE tissue and cfDNA was determined using the Promega MSI Analysis System. The mutation spectrum in the matched cfDNA samples was determined using the Oncomine PanCancer cfTNA Assay. Inflammatory (IFNG) signatures were assessed using the targeted RNASeq panel, Oncomine Immune Response Research Assay (OIRRA). The clonality of TILs was analyzed using NGS. TMB results generated from the targeted panels using only 20ng DNA input were comparable to WES results. Differences in TMB counts between the NGS assays are likely due to differences in genomic coverage and data filtering. MSI-H status and/or mutations in critical DNA repair or proofreading genes such as MSH6 and POLE correlated with high TMB. Gene expression profiling using OIRRA was successful for all FFPE RNA samples. In summary, this study demonstrates that comprehensive genomic analysis of predictive biomarkers for CI therapy can be performed from limited FFPE samples using co-extraction methods and a combination of targeted NGS and PCR-based assays. DNA and RNA quality have a significant impact on the accuracy of TMB analysis and gene expression profiling. In cases where no tumor tissue is available, the analysis of biomarkers such as MSI and mutation signatures may be possible using cfDNA. Citation Format: Xiaodong Wang, Zhenyu Yan, Quyen Vu, David Smith, Chad Galderisi, Cynthia S. Spittle, Jin Li. Comprehensive genomic analysis of predictive biomarkers for checkpoint inhibitor therapy from a limited amount of FFPE tissue and cfDNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1702.

Simultaneous DNA and protein extraction using trypsin

DNA on touched objects is often present in low amounts, causing poor STR typing results. Testing genetically variable proteins is a promising approach for obtaining additional individualization. We are showing here that it is possible to obtain PCR-STR results for DNA and mass spectrometry protein sequences starting with a single trypsin digestion. For a standard DNA extraction method using Millipore Microcon MW100 filter units, proteinase K was replaced with trypsin, and sodium dodecyl sulfate with ProteaseMAX, a mass spectrometry compatible detergent. This neatly separates both substances: DNA is retained by the membrane with digested proteins in the flow through. It was also possible to amplify trypsin extracted DNA without further purification, but Microcon separation of both fractions was more suitable for both STR and mass spectrometry analysis. When tested in parallel to a standard proteinase K method, the Microcon co-extraction method showed better DNA typing success rates. Mass spectrometry for the Microcon based trypsin co-extraction method yielded a large number of identified proteins, including tissue specific proteins for both fingerprint and saliva samples.

Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections

Current extraction methods often extract DNA and RNA separately, and few methods are capable of co-extracting DNA and RNA from sputum. We established a nucleic acid co-extraction method from sputum based on magnetic beads and optimized the method by evaluating influencing factors, such as the guanidinium thiocyanate (GTC) and dithiothreitol (DTT) concentrations, magnetic bead amount, incubation temperature, lysis buffer pH and RNA carrier type. The feasibility of the simultaneous nucleic acid co-extraction method was evaluated by amplifying DNA and RNA viruses from a single clinical specimen with a multiplex RT-qPCR method. Both DNA and RNA were most efficiently extracted when the GTC and DTT concentrations were 2.0 M and 80 mM, respectively, 20 μl magnetic beads were added, the incubation temperature was 80 °C, the pH was 8 or 9, and RNA carrier A was used. Therefore, we established a simple method to extract nucleic acids from two important respiratory viruses compared with other commercial kits. This magnetic beads-based co-extraction method for sputum followed by a multiplex RT-qPCR can rapidly and precisely detect DNA and RNA viruses from a single clinical specimen and has many advantages, such as decreased time, low cost, and a lack of harmful chemicals.

Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses.

Adequate comparisons of DNA and cDNA libraries from complex environments require methods for co-extraction of DNA and RNA due to the inherent heterogeneity of such samples, or risk bias caused by variations in lysis and extraction efficiencies. Still, there are few methods and kits allowing simultaneous extraction of DNA and RNA from the same sample, and the existing ones generally require optimization. The proprietary nature of kit components, however, makes modifications of individual steps in the manufacturer’s recommended procedure difficult. Surprisingly, enzymatic treatments are often performed before purification procedures are complete, which we have identified here as a major problem when seeking efficient genomic DNA removal from RNA extracts. Here, we tested several DNA/RNA co-extraction commercial kits on inhibitor-rich soils, and compared them to a commonly used phenol-chloroform co-extraction method. Since none of the kits/methods co-extracted high-quality nucleic acid material, we optimized the extraction workflow by introducing small but important improvements. In particular, we illustrate the need for extensive purification prior to all enzymatic procedures, with special focus on the DNase digestion step in RNA extraction. These adjustments led to the removal of enzymatic inhibition in RNA extracts and made it possible to reduce genomic DNA to below detectable levels as determined by quantitative PCR. Notably, we confirmed that DNase digestion may not be uniform in replicate extraction reactions, thus the analysis of “representative samples” is insufficient. The modular nature of our workflow protocol allows optimization of individual steps. It also increases focus on additional purification procedures prior to enzymatic processes, in particular DNases, yielding genomic DNA-free RNA extracts suitable for metatranscriptomic analysis.

A 17-month time course study of human RNA and DNA degradation in body fluids under dry and humid environmental conditions.

Blood, saliva, and semen are some of the forensically most relevant biological stains commonly found at crime scenes, which can often be of small size or challenging due to advanced decay. In this context, it is of great importance to possess reliable knowledge about the effects of degradation under different environmental conditions and to use appropriate methods for retrieving maximal information from limited sample amount. In the last decade, RNA analysis has been demonstrated to be a reliable approach identifying the cell or tissue type of an evidentiary body fluid trace. Hence, messenger RNA (mRNA) profiling is going to be implemented into forensic casework to supplement the routinely performed short tandem repeat (STR) analysis, and therefore, the ability to co-isolate RNA and DNA from the same sample is a prerequisite. The objective of this work was to monitor and compare the degradation process of both nucleic acids for human blood, saliva, and semen stains at three different concentrations, exposed to dry and humid conditions during a 17-month time period. This study also addressed the question whether there are relevant differences in the efficiency of automated, magnetic bead-based single DNA or RNA extraction methods compared to a manually performed co-extraction method using silica columns. Our data show that mRNA, especially from blood and semen, can be recovered over the entire time period surveyed without compromising the success of DNA profiling; mRNA analysis indicates to be a robust and reliable technique to identify the biological source of aged stain material. The co-extraction method appears to provide mRNA and DNA of sufficient quantity and quality for all different forensic investigation procedures. Humidity and accompanied mold formation are detrimental to both nucleic acids.

Evaluation of in-tube solid-phase microextraction method for co-extraction of acidic, basic, and neutral drugs

Simultaneous extraction of acidic, basic, and neutral drugs from different biological samples is a considerable and disputable concept in sample preparation strategies.

Co-extraction of DNA and PLFA from soil samples

Lipid/DNA co-extraction from one sample is attractive in limiting biases associated with microbial community analysis from separate extractions. We sought to enhance established co-extraction methods and use high-throughput 16S rRNA sequencing to identify preferentially extracted taxa from co-extracted DNA. Co-extraction results in low DNA yields and distinct community structure changes.

RNA/DNA co-analysis from human skin and contact traces – results of a sixth collaborative EDNAP exercise

The European DNA profiling group (EDNAP) organized a sixth collaborative exercise on RNA/DNA co-analysis for body fluid/tissue identification and STR profiling. The task was to identify skin samples/contact traces using specific RNA biomarkers and test three housekeeping genes for their suitability as reference genes. Eight stains, a skin RNA dilution series and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 22 participating laboratories using RNA extraction or RNA/DNA co-extraction methods. Two sets of previously described skin-specific markers were used: skin1 pentaplex (LCE1C, LCE1D, LCE2D, IL1F7 and CCL27) and skin2 triplex (LOR, KRT9 and CDSN) in conjunction with a housekeeping gene, HKG, triplex (B2M, UBC and UCE). The laboratories used different chemistries and instrumentation. All laboratories were able to successfully isolate and detect mRNA in contact traces (e.g., human skin, palm-, hand- and fingerprints, clothing, car interiors, computer accessories and electronic devices). The simultaneous extraction of RNA and DNA provides an opportunity for positive identification of the tissue source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling. The skin markers LCE1C and LOR and the housekeeping gene marker B2M were detected in the majority of contact traces. Detection of the other markers was inconsistent, possibly due to the low amounts and/or poor quality of the genetic material present in shed skin cells. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid/tissue identification method that can easily be combined with current STR typing technology.