Kit Qiagen Research Articles

Extracellular vesicles are increased in the serum of children with autism spectrum disorder, contain mitochondrial DNA, and stimulate human microglia to secrete IL-1\u03b2

BackgroundAutism spectrum disorder (ASD) has been associated with brain inflammation as indicated by the activation of microglia, but the triggers are not known. Extracellular vesicles (EVs) are secreted from many cells in the blood and other biological fluids and carry molecules that could influence the function of target cells. EVs have been recently implicated in several diseases, but their presence or function in ASD has not been studied.MethodsEVs were isolated from the serum of children with ASD (n = 20, 16 males and 4 females, 4–12 years old) and unrelated age and sex-matched normotypic controls (n = 8, 6 males and 2 females, 4–12 years old) using the exoEasy Qiagen kit. EVs were characterized by determining the CD9 and CD81 membrane-associated markers with Western blot analysis, while their morphology and size were assessed by transmission electron microscopy (TEM). Human microglia SV40 were cultured for 24 h and then stimulated with EVs (1 or 5 μg/mL), quantitated as total EV-associated protein, for 24 or 48 h. IL-1β secretion was measured by ELISA. The results were analyzed using the Mann-Whitney U non-parametric test, and all statistical analyses were performed using Graph Pad Prism 5.ResultsEVs were isolated and shown to be spherical structures (about 100 nm) surrounded by a membrane. Total EV-associated protein was found to be significantly increased (p = 0.02) in patients as compared to normotypic controls. EVs (5 μg/mL) isolated from the serum of patients with ASD stimulated cultured human microglia to secrete significantly more of the pro-inflammatory cytokine interleukin IL-1β (163.5 ± 13.34 pg/mL) as compared to the control (117.7 ± 3.96 pg/mL, p < 0.0001). The amount of mitochondrial DNA (mtDNA7S) contained in EVs from children with ASD was found to be increased (p = 0.046) compared to the normotypic controls.ConclusionsThese findings provide novel information that may help explain what triggers inflammation in the brain of children with ASD and could lead to novel effective treatments.

INVOLVEMENT OF JNK MAPK CASCADES IN THE FORMATION OF ADHESIONS IN THE ABDOMINAL CAVITY

Adhesive process in the abdominal cavity is one of the most frequent complications of intra-abdominal surgery.The aim of the study was to evaluate the activation of the JNK MAPK cascade during the experimental adhesion process.Materials and methods. Experimental studies were performed on 40 male Wistar rats (9 months old). We modeled the adhesive process in the abdominal cavity. At the time from 2 hours (2, 6, 12 hours) to 30 days (1, 3, 7, 14, 30 days), a JNK MAPK cascade was evaluated in the zone of damage to the serosa using immunohistochemical methods (painting on JNK1 Phospho (pT183) / JNK2 Phospho (pT183) / JNK3 Phospho (pT221)) and real-time PCR (the MAP Kinase Signaling Pathway RT2 - Profiler ™ PCR Array (Qiagen) kit). Results. We have found that the expression of the phosphorylated part of the JNK MAPK cascades has an undulating dynamics with two peaks on the 3rd and 14th day. It has been determined that all three JNK cascades are involved in the process, and all JNK cascades are synchronously activated on the 3rd day (JNK1 increases 12-fold in comparison with the group of intact animals, JNK2 is 8-fold, JNK3 is 10-fold). On the 14th day, the activity of the JNK3 cascade is the most intensively increased – 30 times (for JNK1 – about 6 times, JNK2 – 12 times). Conclusion. Significant activation of MARK cascades has been established on the 14th day after traumatic injury, which indicates the need for long-term prevention of posttraumatic changes, in particular, the formation of adhesions.

Abstract 2707: Integrated analysis of colorectal carcinoma by co-extraction of RNA, DNA and protein from FFPE tumor samples

Abstract Background: Integrated analysis of multi-omic data is important in understanding oncogenesis, pathology, and drug mechanism of action. Formalin-fixed, paraffin-embedded (FFPE) tissues comprise the bulk of archival specimens in hospitals and clinical trials. Commercial kits are commonly used to extract RNA, DNA, or protein for biomarker studies; however, high-quality extractions are challenging due to crosslinking. We explored the feasibility of performing proteomic, transcriptomic, and genetic analysis from limited FFPE samples collected in clinical trials through a pilot study in colorectal cancer (CRC) and normal tissue. Methods: Qiagen kits were employed, with some modifications, to extract nucleic acid and protein from FFPE sample lysates that would be amenable to next-generation sequencing and liquid chromatography/mass-spectrometry (LC/MS) proteomic profiling. Commercially sourced FFPE slides from CRC biopsies and normal colon, heart, and skin samples were processed (n = 10 each; ~600-mm2 by 5-μm/slice). RNA-seq library preparation was performed using the Illumina® TruSeq Access kit. Label-free LC/MS proteomic analysis was carried out using trypsin/Lys-C-digested protein fragments on an EASY-nLC™ 1200 coupled to a Q Exactive™ Plus (Thermo Fisher). MS data were processed with MaxQuant to estimate protein abundance (Cox and Mann, Nat Biotech 2008). Results: Of 40 samples tested, 38 yielded quantifiable nucleic acid and protein of sufficient quality for transcriptional and proteomic analyses. Mean RNA yield was ~300 ng (150 ng-1.2 µg). RNA degradation was significant, with a mean DV&amp;gt;200 of 45% (Agilent Bioanalyzer). Mean DNA yield was 530 ng, with a mean fragment size of 2 kb. Mean protein yield was 50 µg (1 µg-300 µg). RNA libraries had a mapping rate range of 85%-90% and a coding rate range of 70%-80%; ~16,000 genes were reliable detected (log2 TPM &amp;gt; 1). The number of unique proteins detected ranged from 3,000 to 4,000 for CRC, normal colon, and heart samples; skin samples averaged 1,000 proteins and had the lowest overall yield. Correlation of RNA and protein expression for the same genes was weak (Spearman's rho ~0.3-0.4) at the per-sample level but statistically significant. Using linear models, we identified differentially expressed transcripts and proteins between the CRC and normal colon samples. Pathway enrichment analysis of both modalities indicated changes in cell cycle, which is consistent with rapid growth of tumor cells. Changes in nucleoside metabolism, extracellular matrix remodeling, and innate immune response were more apparent at the protein level. Conclusion: We found that multi-omic analysis was feasible with FFPE samples, and proteomics can be used to validate RNA results. Additionally, proteomics reveal post-translational events, such as extracellular matrix remodeling, that provide unique insights into cancer pathology. Citation Format: Vishal Patel, Ji Gao, Mingyi Liu, Aiqing He, Xi-Tao Wang, Kathryn Vanderlaag, Ariella Sasson, Stefan Kirov, Sunil Kuppasani, Omar J. Jabado, Kandasamy Ravi, Ashok Dongre, Julie Carman, Heidi LeBlanc. Integrated analysis of colorectal carcinoma by co-extraction of RNA, DNA and protein from FFPE tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2707.

Abstract 2471: Clinical significance of noncoding circHIPK3 RNA in human hepatocellular carcinoma

Abstract Background and Aim: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, with poor prognosis and high risk of recurrence. Previous studies have shown that various microRNAs (miRNAs) are frequently dysregulated in HCC, which contributes to cancer development and progression. Nonetheless, the regulation mechanism of miRNAs is still unclear. Circular noncoding RNA (circRNA) is highly conserved and stable covalently closed RNA circles with gene-regulatory potential. Recently, one abundant circRNA from HIPK3 gene, circHIPK3, has been identified, and its function has been elucidated as multiple miRNAs sponge. Thus, we aimed to investigate the clinical relevance of circHIPK3 in HCC. Materials and Methods: We analyzed clinical specimens from 152 pairs of HCC and corresponding normal liver (NL) tissues. Total RNAs were isolated from clinical tissues using Qiagen kit. CircHIPK3 expression levels were determined by quantitative real-time PCR (qRT-PCR), and its expression was normalized with GAPDH. In addition, we analyzed the correlation between circHIPK3 expression and various clinicopathologic features of HCC patients. Results: CircHIPK3 expression was significantly downregulated in HCC tissues compared to corresponding NL tissues (P=0.036). In HCC patients, circHIPK3 expression was strongly suppressed in more advanced tumors (P&amp;lt;0.001). Further correlation analysis showed that circHIPK3 expression was significantly associated with T stage (P&amp;lt;0.001), TNM stage (P&amp;lt;0.001), BCLC stage (P&amp;lt;0.001), and alpha-fetoprotein (AFP) expression (P=0.033). Conclusions: We have determined clinical significances of circHIPK3 expression in HCC, which may provide clinical evidence for the potential of circHIPK3 as novel markers for diagnosis and predicting prognosis in HCC patients. Citation Format: Gyeonghwa Kim, Jun Sik Yoon, Se Young Jang, Soo Young Park, Won Young Tak, Young-Oh Kweon, Keun Hur. Clinical significance of noncoding circHIPK3 RNA in human hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2471.

Abstract 3655: DNA methylation analysis in liquid biopsy: A detailed study on quality control, sample storage and whole genome amplification procedures for downstream applications

Abstract Background: Studies on epigenetic alterations and especially DNA methylation in liquid biopsy samples like plasma circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) are of growing interest nowadays. To ensure the reliability of DNA methylation results prior to any clinical applications in liquid biopsy, there is an urgent need to standardize a plethora of parameters. The aim of our study was to evaluate: a) the stability of DNA methylation in plasma, b) the stability of sodium bisulfite (SB) converted DNA samples under different storage temperatures, and c) the accuracy and precision of whole genome amplification (WGA) protocols for downstream DNA methylation analysis. Methods: a) Stability of DNA methylation in plasma: Plasma from healthy donors was pooled and aliquoted in six tubes and spiked with genomic DNA (gDNA) isolated from 106 MCF-7 cells. Five aliquots were kept at -700C, while one was immediately processed for ctDNA isolation and SB treatment. Stored aliquots were processed in the same way after storage for a period of 3 months. All SB-treated DNA samples were further evaluated by real time methylation specific PCR (MSP) for ACTB and SOX17. b) Stability of SB-treated DNA: gDNA was isolated from 106 SKBR3 cells and processed for downstream SB-treatment. SB-treated DNA was split into aliquots and stored at -20°C and -70°C. One aliquot, used as control, was immediately processed for SB treatment followed by real time MSP for ACTB and BRMS1 amplification. The remaining aliquots were processed in exactly the same way after storage for different time points with a maximum period of one year. Additionally, a sample that was subjected to repeated freeze-thaw cycles was added in each run. c) Evaluation of WGA for SB-treated DNA: we evaluated two different SB conversion kits, EZ DNA Methylation-Gold (Zymo), and Epitect Fast Bisulfite, (Qiagen) in combination with downstream WGA (EpiTect Whole Bisulfitome Kit, Qiagen), using 0%, 100% methylated DNA standards and MCF-7, SKBR3 cell lines. Results: a) DNA methylation in plasma is stable at -700C for at least 3 months, since the average Cqs detected, varied from 1 to 2 cycles for ACTB and SOX17, respectively, b) Quality of SB-treated DNA is not affected by repeated freezing-thawing cycles; SB-treated DNA samples are stable when kept for up to one year both at -200C and -700C. c) Identical results by real time MSP were obtained before and after WGA amplification (Qiagen), after using either the Zymo or Qiagen kits for SB conversion. Conclusions: Plasma and SB-treated DNA samples are stable and can be safely used for multiple real time MSP assays over time when kept at -70°C. Downstream WGA of SB-treated DNA could bypass the limited amount of available sample for liquid biopsy applications. In any case, quality control checking should be included in each step of analysis. Citation Format: Sofia Mastoraki, Maria Chimonidou, Eleni Tzanikou, Evi S. Lianidou. DNA methylation analysis in liquid biopsy: A detailed study on quality control, sample storage and whole genome amplification procedures for downstream applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3655.

Comparison of RNA extraction methods for the detection of BNYVV rhizomania virus from roots of sugar beet

Rhizomania is one of serious threat to sugar beet production in Morocco and in several parts of the world. This disease led to a statistically significant decrease in the quality and yield of sugar beet plantations. Therefore, this study aimed at comparing the efficacy of six commonly used RNA extraction methods for the detection, recovery of RNA of beet necrotic yellow vein virus (BNYVV) and removal of amplification inhibitors by reverse transcription-polymerase chain reaction (RT-PCR). The efficiency of these extraction methods was then compared to that of a commercial isolation kit with high content of phenolic compounds. The results showed that the extraction with the lithium chloride technique, the commercial kit, and direct and membrane spotting crude extract methods were found effective in yielding a higher purity and a higher concentration of RNA when compared to the other tested methods. Extraction with the lithium chloride technique and the Qiagen kit (RNeasy Plant Mini Kit) allowed the most intense band, whereas the CTAB method has generated the least intense band. Furthermore, the silica capture extraction method did not yield any RNA after extraction and electrophoresis. Consequently, it was concluded that, of these six methods, the lithium chloride technique and the Qiagen kit are the most appropriate for the extraction of viral RNA from sugar beet samples prior to RT-PCR for detecting BNYVV.

The Importance of New Generation Sequencing (NGS) HLA Typing in Renal Transplantation—Preliminary Report

IntroductionThanks to new generation sequencing (NGS) and expansion of HLA typing with additional loci, it will be possible to increase the effectiveness of graft survival and to avoid complications related to the immune system. New pharmacogenetic factors are still being researched to develop better immunosuppressive treatment. Material and methodsThe incidence of polymorphic HLA loci variants was established, based on a high-resolution NGS method in kidney graft recipients. Furthermore, haplotypic analysis between examined loci was conducted to type additional loci that may influence the transplantation result. A total of 120 kidney recipients were enrolled in the study. A commercial DNA extraction kit in Tubes (QIAamp DNA Blood Mini Kit Qiagen, Germany) was used to isolate DNA from the blood. Sequencing library preparation was done with TruSight HLA set. The Conexio computer program was used to analyse the results of HLA typing. ResultsThe patients with alleles A*02:01:01, B*44:02:01, C*03:03:01, C*01:02:01, C*05:01:01, C*07:02:01, DQB1*03:03:02, DQB1*06:04:01, or with haplotypic variation A*25:01:01-B*18:01:01- C*15:01:01 were taking the highest doses of cyclosporine (CsA), in contrast to patients with allele B*18:01:01, DQB1*06:02:01, DQB1*02:02:01, or haplotypic variation A*02:01:01- B*44:02:01-C*01:01:01, who were taking the lowest doses. The highest dose of tacrolimus (TAC) was administered to patients with alleles A*68:01:02, A*29:01:01, B*07:02:01, B*35:02:01, B*38:01:01, DRB1*12:01:01, DQB1*05:03:01, or haplotypic variations A*02:01:01-B*57:01:01-C*07:01:01, A*03:01:01-B*07:02:01-C*13:01:01, A*29:02:01-B*44:03:01- C*07:01:01, and A*01:01:01-B*08:01:01-C*03:01:01. Additionally, it was established that HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DPA1, and HLA-DQA1 show very slight polymorphism, which suggests that there is no need for their typing for transplantation purposes. Moreover, loci HLA-C, HLA-DQB1, and HLA-DPB1, which are not routinely examined in recipient-donor matching, show genetic variability that may increase the risk of transplant rejection or shortened graft life. ConclusionsExpanding the qualification procedure to include allele genotyping could allow clinicians to establish immunosuppressive treatment schemes that would be optimally suited for recipients' phenotype.

Evaluation of Genomic DNA-Extraction Methods for Molecular Analysis of Solanecio biafrae

ABSTRACT Solanecio biafrae (Oliv. & Hiern) C. Jеffrеу, an indigenous vegetable of the humid tropics, is rich in minerals and easy to cultivate, but its vegetative growth is slow, making production fall short of demand. An efficient and effective genomic DNA-extraction method is necessary for a comprehensive molecular analysis to increase leaf yield and further improve proximate and mineral contents of the vegetable. Cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), and Qiagen kit (QIAGEN) methods were evaluated for extraction of genomic DNA from young leaves suitable for PCR amplification for molecular analyses. Quantity and quality of the DNA were examined spectrophotometerically and with gel electrophoresis. Amplification of the DNA by PCR for molecular analysis was assessed with five inter-simple sequence repeat (ISSR) primers. The mean DNA yield using QIAGEN, CTAB, and SDS methods were 71.0, 35.4, and 38.8 µg∙µL−1∙g−1 of tissue, respectively. The mean 260/280 nm ratios of QIAGEN, CTAB, and SDS were 1.84, 1.83, and 1.79, respectively. Gel electrophoresis indicated presence of DNA free of contaminants in all samples using three methods. A total of 270 distinct bands were produced by five ISSR primers with similar patterns among three DNA-extraction methods, ranging from 200–2900 base pairs. Three methods are capable of extracting adequate genomic DNA suitable for PCR amplification. The QIAGEN method produced the highest DNA yield, which was of the same quality with DNA obtained using CTAB and SDS methods.

Ability of Polyphosphate and Nucleic Acids to Trigger Blood Clotting: Some Observations and Caveats.

Polyphosphate plays several roles in coagulation and inflammation, while extracellular DNA and RNA are implicated in thrombosis and as disease biomarkers. We sought to compare the procoagulant activities of polyphosphate versus DNA or RNA isolated from mammalian cells. In a recent study, we found that much of the procoagulant activity of DNA isolated from mammalian cells using Qiagen kits resisted digestion with nuclease or polyphosphatase, and even resisted boiling in acid. These kits employ spin columns packed with silica, which is highly procoagulant. Indeed, much of the apparent procoagulant activity of cellular DNA isolated with such kits was attributable to silica particles shed by the spin columns. Therefore, silica-based methods for isolating nucleic acids or polyphosphate from mammalian cells are not suitable for studying their procoagulant activities. We now report that polyphosphate readily co-purified with DNA and RNA using several popular isolation methods, including phenol/chloroform extraction. Thus, cell-derived nucleic acids are also subject to contamination with traces of cellular polyphosphate, which can be eliminated by alkaline phosphatase digestion. We further report that long-chain polyphosphate was orders of magnitude more potent than cell-derived DNA (purified via phenol/chloroform extraction) or RNA at triggering clotting. Additional experiments using RNA homopolymers found that polyG and polyI have procoagulant activity similar to polyphosphate, while polyA and polyC are not procoagulant. Thus, the procoagulant activity of RNA is rather highly dependent on base composition.

Potential Value of Qiagen and PrepIT•MAX Kits in Extraction of Mycobacterial DNA From Presumptive Tuberculosis Archived Formalin-Fixed Paraffin-Embedded Tissues

DNA analysis has potential for screening for and diagnosing a variety of conditions as well as the characterization of various pathogens for many purposes including to identify genetic disorders and mutations, study genetic diversity, and establish evolutional trends.Our study compared the performance of 2 DNA extraction kits: Qiagen and prepIT•MAX. The study tested 160 formalin-fixed paraffin-embedded (FFPE) human tissue samples that had been collected at Muhimbili National Hospital (MNH) between 2010 and 2016. For each sample, DNA extraction was performed using both the Qiagen and prepIT•MAX kits followed by polymerase chain reaction (PCR) tests to target the RNA polymerase gene and gel electrophoresis.The findings showed that the Qiagen was 3 times superior to the prepIT•MAX kit in successfully extracting mycobacterial DNA from presumptive tuberculosis (TB) FFPE tissues. Of the 160 previously Ziehl-Neelsen stain-negative Mycobacterium tuberculosis suspicious tissue samples, 12 (7.5%) tested positive with the PCR. Of the 12 PCR-detected positive samples, 8 (66.7%) yielded positive results with the Qiagen kit only and 4 (33.3%) yielded positive results with both Qiagen and prepIT•MAX kits. Additionally, 10 (83.3%) came from well-formed granuloma, 1 (8%) from caseous necrosis, and 1 (8.3%) Langhans-type giant cells endorsing their potential for housing infection such as TB adenitis.A combination of molecular techniques, microscopy, and pathological features increases detection of M. tuberculosis from FFPE tissues. Both the Qiagen and the prepIT•MAX DNA extraction kits have shown a remarkable capability for extracting DNA from M. tuberculosis, although examination of FFPE tissues is not an intended use for the prepIT MAX, according to the manufacturer. In resource-limited countries, however, these kits may complement each other. We recommend further studies for validation and optimization, which includes the cost effectiveness of prepIT•MAX extraction kit to advocate for its use in extraction of mycobacterial DNA from FFPE tissues.

KRAS and NRAS Testing in Metastatic Colorectal Cancer in Central Iran (Tehran): A Review on Literature of the Middle East

Abstract Context: The incidence of colorectal cancer (CRC) in the past three decades in Iran has made it as a major public health burden. Aims: The aim of this study is to report the prevalence of KRAS and NRAS mutations in Iran and the correlation between KRAS mutation status with clinicopathological factors and survival.Materials and Methods: In a cross-sectional study, 144 patients were entered into the study based on the criteria. Age, sex, tumor site, grade, metastasis location, familial history, KRAS/NRAS status, and survival were checked for all patients, and the patients were followed for 1 year. DNA was extracted with FFPE QIAGEN kit and then polymerase chain reaction for amplification of gene segments of KRAS and NRAS genes. Results: The mean age at diagnosis was 52.9 years (range: 27–72 years) that 39.6% patients had age &lt;50 years and 54.2% were men. KRAS mutation was significantly more in the patients with age ≥50 compared with KRAS wild type. Furthermore, the 6-month overall survival rate in KRAS mutation patients was significantly more than KRAS wild-type patients. Liver metastasis (72.9%) had the highest prevalence of metastasis in the patients, and Grade II with 64.6% had the most prevalence. Conclusions: The metastatic CRC was more prevalent in men than women, and the mean age varied around 50–60 years. The results showed that the present study had the highest prevalence of KRAS mutation in the Middle East and Pakistan with the lowest prevalence in CRC patients.

Methodology challenges in studying human gut microbiota \u2013 effects of collection, storage, DNA extraction and next generation sequencing technologies

The information on microbiota composition in the human gastrointestinal tract predominantly originates from the analyses of human faeces by application of next generation sequencing (NGS). However, the detected composition of the faecal bacterial community can be affected by various factors including experimental design and procedures. This study evaluated the performance of different protocols for collection and storage of faecal samples (native and OMNIgene.GUT system) and bacterial DNA extraction (MP Biomedicals, QIAGEN and MO BIO kits), using two NGS platforms for 16S rRNA gene sequencing (Ilumina MiSeq and Ion Torrent PGM). OMNIgene.GUT proved as a reliable and convenient system for collection and storage of faecal samples although favouring Sutterella genus. MP provided superior DNA yield and quality, MO BIO depleted Gram positive organisms while using QIAGEN with OMNIgene.GUT resulted in greatest variability compared to other two kits. MiSeq and IT platforms in their supplier recommended setups provided comparable reproducibility of donor faecal microbiota. The differences included higher diversity observed with MiSeq and increased capacity of MiSeq to detect Akkermansia muciniphila, [Odoribacteraceae], Erysipelotrichaceae and Ruminococcaceae (primarily Faecalibacterium prausnitzii). The results of our study could assist the investigators using NGS technologies to make informed decisions on appropriate tools for their experimental pipelines.

Potential Value of Qiagen and PrepIT•MAX Kits in Extraction of Mycobacterial DNA From Presumptive Tuberculosis Archived Formalin-Fixed Paraffin-Embedded Tissues

Background:DNA analysis has potential for screening for and diagnosing a variety of conditions as well as the characterization of various pathogens for many purposes including to identify genetic disorders and mutations, study genetic diversity, and establish evolutional trends.Methods:Our study compared the performance of 2 DNA extraction kits: Qiagen and prepIT•MAX. The study tested 160 formalin-fixed paraffin-embedded (FFPE) human tissue samples that had been collected at Muhimbili National Hospital (MNH) between 2010 and 2016. For each sample, DNA extraction was performed using both the Qiagen and prepIT•MAX kits followed by polymerase chain reaction (PCR) tests to target the RNA polymerase gene and gel electrophoresis.Results:The findings showed that the Qiagen was 3 times superior to the prepIT•MAX kit in successfully extracting mycobacterial DNA from presumptive tuberculosis (TB) FFPE tissues. Of the 160 previously Ziehl-Neelsen stain-negative Mycobacterium tuberculosis suspicious tissue samples, 12 (7.5%) tested positive with the PCR. Of the 12 PCR-detected positive samples, 8 (66.7%) yielded positive results with the Qiagen kit only and 4 (33.3%) yielded positive results with both Qiagen and prepIT•MAX kits. Additionally, 10 (83.3%) came from well-formed granuloma, 1 (8%) from caseous necrosis, and 1 (8.3%) Langhans-type giant cells endorsing their potential for housing infection such as TB adenitis.Conclusions:A combination of molecular techniques, microscopy, and pathological features increases detection of M. tuberculosis from FFPE tissues. Both the Qiagen and the prepIT•MAX DNA extraction kits have shown a remarkable capability for extracting DNA from M. tuberculosis, although examination of FFPE tissues is not an intended use for the prepIT MAX, according to the manufacturer. In resource-limited countries, however, these kits may complement each other. We recommend further studies for validation and optimization, which includes the cost effectiveness of prepIT•MAX extraction kit to advocate for its use in extraction of mycobacterial DNA from FFPE tissues.

HLA-C*17, DQB1*03:01, DQA1*01:03 and DQA1*05:05 Alleles Associated to Bullous Pemphigoid in Brazilian Population.

BackgroundBullous pemphigoid (BP) is an autoimmune disease with bullous vesicles and an incidence of 0.2 to 1.4 per 100,000 inhabitants. Many studies have been published demonstrating the association of pemphigoid with HLA class II system alleles in different populations, however there are no data on the BP, one of the most heterogeneous in the world.ObjectiveTo typify HLA alleles in Brazilians with Bullous pemphigoid.MethodsThe study group included 17 Brazilian patients with a confirmed diagnosis of BP from a hospital in Sao Paulo city, southeast Brazil. DNA was extracted from peripheral blood using Qiagen kits and HLA A, B, C, DR and DQ typing was performed using polymerase chain reaction. The control group was composed of a database of 297 deceased donors from the city of Sao Paulo. The statistical significance level was adjusted using the Bonferroni correction depending on the phenotypic frequencies evaluated for HLA class I (A, B and C) and class II (DRB1, DQB1 and DQA1).ResultsOur findings show that alleles HLA C*17, DQB1*03:01, DQA1*01:03 and DQA1*05:05 are associated with the onset of the disease in the Brazilian population, with relative risks of 8.31 (2.46 to 28.16), 3.76 (1.81 to 7.79), 3.57 (1.53 to 8.33), and 4.02 (1.87 to 8.64), respectively (p<0.005).ConclusionOur data indicate that Brazilian patients with BP present the same genetic predisposition linked to HLA-DQB1*03:01 previously reported in Caucasian and Iranian individuals and our study introduces three new alleles (C*17, DQA1*01:03 and DQA1*05:05) involved in the pathophysiology of BP.

Effect of selected polymorphisms of genes lep, MTHFR and FTO to BMI level and gender-specificity

The aim of this study is to investigate the effect of selected polymorphisms LEP (G2548A), MTHFR (C677T) and FTO (rs1121980) on body mass index in humans. In the study participated 79 people from Slovakia with some genetic relatedness. Genomic DNA was isolated from the buccal swabs using a commercial kit Qiagen DNA Mini Kit (Qiagen). The detection of SNP polymorphisms in human genes LEP, MTHFR and FTO was performed using molecular genetics methods such as PCR-RFLP and ARMS. The most common genotypes in all 3 polymorphism were found in heterozygous form (for LEP AG = 0.5190, for MTHFR CT = 0.519, for FTO CT =0.4051). The LEP gene had increased frequency of G allele (0.5506), the MTHFR gene T allele (0.6646) and FTO gene T allele (0.50635). The least frequent genotype in LEP was AA (0.1899), in MTHFR was TT (0.0759), in FTO it was CC (0.2911). According to the results we can assume that the genotypes AA (LEP G2548A), CC (FTO rs1121980) and TT (MTHFR C677T) in case of women have a protective effect on the prevalence of obesity, based on BMI, compared to the other genotypes and this polymorphism is gender-specific. Added anthropometric measurements, blood test and extension of the group in the future evaluation could increase the statistical relevance in relation to obesity and gender-specificity.

Evaluation of two RNA extraction methods in children's saliva

ABSTRACT Objective To standardize an RNA (ribonucleic acid) extraction protocol in children's saliva specimens. Methods The study was conducted on saliva specimens from 60 children who participated in the study with their parents’ authorization. Comparison of two RNA extraction methods was established; methods assessed concentration, quality and yield. Moreover, genic expression of GUSB gene was achieved with RT-PCR (reverse transcriptasepolymerase chain reaction). Data were analyzed through measurements of central tendency and dispersion, frequencies and percentages. T-Student and χ2 tests were used in order to differentiate between both methods (p Results Analysis of cell amounts per saliva ml revealed a mean of 564,977.8 (SD = 246,678.6); a RNA in children's saliva extraction method was standardized, specifically the method using RNeasy® Protect Saliva Mini Kit Qiagen exhibited better characteristics of RNA concentration (p = 0.0000) and yield (p = 0.0000) when compared to the method using QIAzol®; no RNA (p = 0.146) quality differences were found. Conclusions RNA could be extracted from children's saliva specimens, therefore, it is suggested to use saliva for the molecular analysis of different oral and systemic diseases.

Bacterial community in ancient permafrost alluvium at the Mammoth Mountain (Eastern Siberia)

Permanently frozen (approx. 3.5Ma) alluvial Neogene sediments exposed in the Aldan river valley at the Mammoth Mountain (Eastern Siberia) are unique, ancient, and poorly studied permafrost environments. So far, the structure of the indigenous bacterial community has remained unknown. Use of 16S metagenomic analysis with total DNA isolation using DNA Spin Kit for Soil (MO-Bio) and QIAamp DNA Stool Mini Kit (Qiagen) has revealed the major and minor bacterial lineages in the permafrost alluvium sediments. In sum, 61 Operational Taxonomic Units (OTUs) with 31,239 reads (Qiagen kit) and 15,404 reads (Mo-Bio kit) could be assigned to the known taxa. Only three phyla, Bacteroidetes, Proteobacteria and Firmicutes, comprised >5% of the OTUs abundance and accounted for 99% of the total reads. OTUs pertaining to the top families (Chitinophagaceae, Caulobacteraceae, Sphingomonadaceae, Bradyrhizobiaceae, Halomonadaceae) held >90% of reads. The abundance of Actinobacteria was less (0.7%), whereas members of other phyla (Deinococcus-Thermus, Cyanobacteria/Chloroplast, Fusobacteria, and Acidobacteria) constituted a minor fraction of reads. The bacterial community in the studied ancient alluvium differs from other permafrost sediments, mainly by predominance of Bacteroidetes (>52%). The diversity of this preserved bacterial community has the potential to cause effects unknown if prompted to thaw and spread with changing climate. Therefore, this study elicits further reason to study how reintroduction of these ancient bacteria could affect the surrounding ecosystem, including current bacterial species.

Evaluation of different methods for DNA extraction from human burnt bones and the generation of genetic profiles for identification.

Bone exposure to heat in the presence of moisture breaks the phosphodiester bonds of the backbone, leaving sheared DNA in bone cells. This also limits the possibility of generating a complete profile of the victim. With the increasing incidence of fire outbreaks over the past few years, a paradigm shift to establish identity has been observed, from morphological identification of victims to STR profiling. For this study, 10 bone samples were taken from burnt human bodies that were recovered from different fire outbreak scenes. The DNA from these burnt human tissues was isolated using four different extraction methods: the organic extraction method, the total demineralisation method, the Qiagen kit method, and the Chelex extraction method. STR profiles of victims were generated on a genetic analyser using an AmpFlSTR Identifiler® Plus Kit and analysed on Gene Mapper ID-X. DNA isolated from bones using the total demineralisation extraction method and organic extraction method was of the highest quality due to the efficient removal of inhibitors. DNA obtained using these two methods successfully generated the STR profiles of the victims. The quality of isolated DNA obtained through the Qiagen kit was comparatively low, but STR profiles of the victims were successfully generated. The Chelex kit failed to extract good quality DNA of high quantity from the burnt bones, encountering inhibition in all samples at varying degrees. This study concludes that total demineralisation and the Qiagen kit are sophisticated and reliable methods to obtain a good yield of DNA from burnt human bones, which can be used for the identification of victims.

Abstract 4423: Deep sequencing of lung cancer samples using different library preparation methods produces discordant short non-coding RNA profiles

Abstract Background: RNA-seq is the new standard for profiling coding and non-coding RNAs in a cell. This study compared two methods for miRNA library preparation from NEB and Qiagen. Our goal was to evaluate overall performance in terms of coverage, known miRNA detection, novel miRNA detection, isomiR detection, and tRNA fragment (tRF) detection. Methods: We performed short RNA-seq on 4 lung cancer tumor-normal pairs. We used two library preparation kits: NEB Next Small RNA kit and Qiagen Qiaseq. All 16 preparations were sequenced on an Illumina NextSeq 550 using NextSeq High V2 chemistry and 75 bp single-end reads were obtained. After adapter removal and quality trimming reads were mapped to the hg19 human genome assembly using the Bowtie2 and SHRiMP packages. Results: On average read yield was 46 million reads. A significantly higher portion of the sequenced reads survived adapter removal and quality trimming in the NEB preparations (96.5%) compared to the Qiagen (72.9%). The portion that could be uniquely mapped to hg19 was lower for the NEB kit (41.1%) than for the Qiagen (50.8%).NEB performed better (39.6%) compared with Qiagen (36.9%). Qiagen preparations had lengths between 20 and 25 bp whereas more than half of the mapped reads in the NEB preparation were 26 bp or longer. At the miRNA arm level the Qiagen kit detected significantly more known miRNAs (792) with 10 or more reads, compared with NEB (576). The same 530 miRNA arms were identified by both kits. There was no discernible pattern in the ID’s or the sequence composition of the miRNAs that were identified by each kit. The detection of novel miRNAs was also higher with Qiagen (102), compared with NEB (82). The same 79 novel miRNAs were identified by both. The Qiagen kit detected nearly twice as many isomiRs (5,316) with 10 or more reads compared to NEB (2,958). The same 2,631 isomiRs were identified by both kits. However, isomiR detection across samples was more consistent with the NEB kit. When we computed pairwise Pearson correlations of normal samples, using the most highly expressed miRNAs in each sample, the NEB kit exhibited higher consistency (0.98) compared with the Qiagen kit (0.95). Pearson correlations of tumor samples showed even higher consistency for NEB (0.96) than Qiagen (0.89). Unsurprisingly, Pearson correlations of like samples across the NEB and Qiagen kits was very low: 0.42 for normal and 0.56 for tumor samples. RIN value did not seem to affect the overall performance of either kit. Lastly, we compared tRFs. Here, the differences were very pronounced. For multiple choices of the support threshold the NEB and Qiagen profiles agreed on approximately 33% of the reported tRFs. Conclusions: Library preparation kits give rise to both consistent and divergent results. End users interested in the detection of miRNAs, isomiRs or tRFs may derive greater utility by selecting one kit over another. Citation Format: Brid M. Ryan, Phillipe Loher, Khadijah Mitchell, Adriana Zingone, Yongmei Zhao, Jyoti Shetty, Bao Tran, Isidore Rigoutsos. Deep sequencing of lung cancer samples using different library preparation methods produces discordant short non-coding RNA profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4423. doi:10.1158/1538-7445.AM2017-4423

Estandarización y validación de la prueba de pcr anidada para el diagnóstico de especies del género xyleborus (coleoptera: curculionidae: scolytinae)

En este estudio se estandarizó y validó la técnica de PCR anidada para la detección rápida, sensible y confiable de especies del género Xyleborus mediante el uso de los primers externos CI-J-2183 y TL2-N-3014 e internos J2210 y N2739, que amplifican una banda de 500 pb de la región del gen mitocondrial Citocromo Oxidasa subunidad 1(CO1). Asimismo, se realizó la extracción de ADN de 26 ejemplares de Xyleborus con el kit Qiagen DNeasy® mericom Food (DMF), no reportado previamente su uso para su aplicación en insectos, que resultó en ADN suficiente y de alta calidad para reacciones de amplificación por PCR. El método permitió procesar un solo insecto por extracción, y obtener material genético de muestras conservadas en alcohol de hasta ocho años de antigüedad. El límite de detección se definió hasta una concentración de 780 pg/ul. Se optimizóla PCR en un volumen final de 15 uL sin comprometer calidad de la amplificación. La técnica estandarizada permitió la obtención de ADN de calidad, lo que aseguró alta reproducibilidad y sensibilidad en la detección de especies de Xyleborus y la secuenciación parcial del gen CO1 para las siete especies estudiadas; las secuencias consenso fueron analizadas por homología y depositadas en el GenBank.