High Pure PCR Template Preparation Research Articles

A comparison of five methods to maximize RNA and DNA isolation yield from adipose tissue.

Adipose tissue in the human body occurs in various forms with different functions. It is an energy store, a complex endocrine organ, and a source of cells used in medicine. Many molecular analyses require the isolation of nucleic acids, which can cause some difficulties connected with the large amount of lipids in adipocytes. Ribonucleic acid isolation is particularly challenging due to its low stability and easy degradation by ribonucleases. The study aimed to compare and evaluate five RNA and DNA isolation methods from adipose tissue. The tested material was subcutaneous porcine adipose tissue subjected to different homogenization methods and RNA or DNA purification. A mortar and liquid nitrogen or ceramic beads were used for homogenization. The organic extraction (TriPure Reagent), spin columns with silica-membrane (RNeasy Mini Kit or High Pure PCR Template Preparation Kit), and the automatic MagNA Pure system were used for the purification. Five combinations were compared for RNA and DNA isolation. Obtained samples were evaluated for quantity and quality. The methods were compared in terms of yield (according to tissue mass), purity (A260/280 and A260/230), and nucleic acid degradation (RNA Integrity Number, RIN; DNA Integrity Number, DIN). The results were analyzed statistically. The average RNA yield was highest in method I, which used homogenization with ceramic beads and organic extraction. Low RNA concentration didn't allow us to measure degradation for all samples in method III (homogenization with ceramic beads and spin-column purification). The highest RNA quality was achieved with method IV using homogenization in liquid nitrogen and spin column purification, which makes it the most effective for RNA isolation from adipose tissue. Required values of DNA yield, purity, and integrity were achieved only with spin column-based methods (III and IV). The most effective method for DNA isolation from adipose tissue is method III, using spin-columns without additional homogenization.

First report of grapevine Flavescence dorée phytoplasma in the Czech Republic.

Viticulture is a traditional branch of agriculture in the Czech Republic. Grapevines (Vitis vinifera L.) are cultivated on more than 18,000 hectares in the wine-growing regions of Bohemia and South Moravia. South Moravia alone accounts for more than 90 % of the total wine-growing area in the country. Grapevine yellows are a complex of diseases associated with the phytoplasma presence. Phytoplasmas of at least five different groups can cause similar symptoms in grapevines, and they can be distinguished only on a molecular basis (EPPO 2016). One of them, the grapevine Flavescence dorée phytoplasma (GFDP), which belongs to the 16SrV group, is listed in Annex II, Part B, of the Commission Implementing Regulation (EU) 2019/2072 of 28 November 2019 as a Union quarantine pest known to occur in the Union territory. Official surveys for GFDP in the Czech Republic have been carried out since 2007. In 2016, the first occurrence of Scaphoideus titanus Ball, 1932, the main vector of GFDP, was reported in the South Moravian Region (EPPO Reporting Service 2016). This is a matter of concern because it indicates that there is a risk of disease dissemination to other geographical locations. In September 2021, a total of 250 samples of V. vinifera (preferentially focused on symptomatic plants) and four samples of the wild plant host Clematis vitalba L. were collected from 50 vineyards in South Moravia. Total DNA was extracted using High Pure PCR Template Preparation Kit (Roche, Basel, Switzerland). For phytoplasma screening, a real-time PCR test for generic detection of phytoplasmas was used (Christensen et al. 2004). Samples evaluated as positive were further tested by PCR using phytoplasma universal P1 and P7 primers (Deng and Hiruki 1991; Schneider et al. 1995), followed by nested PCR using the 16SrV group-specific primers fB1 and rULWS1 (Smart et al. 1996). For identification of 16SrV phytoplasma, sequence analysis of the secY-map genetic locus was performed. Two sets of primers were used: FD9f5/MAPr1 primers for the first PCR and FD9f6/MAPr2 for the nested PCR (Arnaud et al. 2007) with PCRBIO TaqMix (PCR Biosystems, London, UK). The nested PCR products were purified and sequenced (Eurofins Genomics, Ebersberg, Germany). The sequences were compared with sequences from the GenBank database. Phytoplasma of the 16SrV group was detected in three samples: V. vinifera cv. Gewürztraminer with symptoms of leaf reddening with no rolling and no other typical symptoms; C. vitalba L. with leaf curling (Fig. 1A); symptomless C. vitalba. The obtained sequences of the secY-map locus of all three 16SrV-positive samples were identical to the sequence of GFDP, isolate Vv-SI257 (Acc. No. FN811141), detected in grapevine in Tuscany (Italy), which belongs to 16SrV group. The sequence of the V. vinifera cv. Gewürztraminer isolate was submitted to GenBank under Acc. No. OQ185203. This isolate belongs to the Map-FD3 cluster (Fig. 1B), and the genotype identified is M51 (corresponding to FD-C), which has already been found in C. vitalba and outbreaks of Flavescence dorée in grapevines in some other European countries (Malembic-Maher et al. 2020). Based on the abovementioned results, this is the first report of the GFDP in the Czech Republic.

B-370 Clinical Utility of microRNA Quantitative Analysis Using Double Quencher Probes and Methylation Analysis by Methylation Sensitive Restriction Enzyme Polymerase Chain Reaction for Bladder Cancer

Abstract Background Histone modifications, DNA methylation, and microRNA (miRNA)-mediated gene silence are examples of epigenetic changes that regulate gene expression without causing sequence changes. The early phases of neoplastic transformation are characterized by the occurrence of these epigenetic changes, which are believed to be crucial for the development and progression of cancer. To increase the sensitivity and practicality of miRNA analysis, we developed a measuring technique using a ZEN double quencher into hydrolysis probe in reverse transcription quantitative polymerase chain reaction (RT-qPCR). Additionally, we developed a method for DNA methylation analysis employing methylation-sensitive restriction enzymes (MSRE) and methylation-insensitive restriction enzymes (MIRE) that can analyze a number of samples quickly. We examined the clinical usefulness of using these systems to understand the level of miRNA expression and methylation in bladder cancer. Methods In this study, 64 biopsied tissues from transurethral bladder tumor resection (TURBT) were used. For miRNA extraction, Cica geniusTM RNA Prep Kit (For Tissue) II (KANTO CHEMICAL CO.,INC.) and a solution that improves miRNA extraction efficiency were used. Using stem-loop RT primers, complementary DNA was created from the isolated miRNA solution. Quantitative analysis was performed by RT-qPCR using a ZEN double quencher probe and miR-21,30a, 200c, and U6 were used as target. Genomic DNA (gDNA) was extracted from the TURBT sample using High Pure PCR Template Preparation Kit (Roche). Restriction enzyme digestion was performed using HapII (Takara Bio) as MSRE and MspI (Takara Bio) as MIRE; the latter has the same recognition site as HapII but is not affected by the presence or absence of methylation. Real-time PCR was performed on the LightCycler 96 (Roche) using Universal ProbeLibrary probes (Roche) targeting ESM1, SFMBT2, and SEPTIN9. From the obtained Cp value, each methylation rate was calculated. The Fisher’s exact test was used to statistically analyze the relationship among miRNA, methylation data and other clinicopathological factors. Results For miR-30a, we observed a higher expression of miRNA was linked to more advanced T stage (P = 0.047). Additionally, ESM1 methylation exhibited a significant correlation with greater grade (P = 0.004) and T stage progress (P = 0.018). Data on miRNA abundance and methylation rate were combined in order to uncover a link with clinical data that was not apparent from either set of data separately. Samples with higher values for both miR-21 and ESM1 methylation rates were shown to have high grade (P = 0.007) and progress to the T stage (P = 0.006). Furthermore, samples with higher values for both miR-200c and SEPTIN9 methylation rates did not progress to the T stage (P = 0.013). Conclusion The combined miRNA and methylation data revealed relationships with clinical results that could not be discovered with either data separately, highlighting the clinical usefulness of the current study’s findings.

Investigation of Leishmania RNA Virus 2 in Leishmania major and Leishmania tropica Strains Isolated from Cutaneous Leishmaniasis Patients in Türkiye

Leishmania RNA virus (LRV) is a double-stranded RNA (dsRNA) virus that is thought to contribute to the severe inflammatory response of the causative Leishmania parasite in the mammalian host by being present in many isolates of Leishmania spp. In our study, it was aimed to obtain data on the presence of Leishmania RNA Virus 2 (LRV2), which is thought to cause a change in the clinical course of leishmaniasis, in Leishmania major and Leishmania tropica isolates isolated from cutaneous leishmaniasis (CL) patients in Türkiye. Leishmania strains stored in liquid nitrogen tank by cryopreservation in Manisa Celal Bayar University Faculty of Medicine Parasite Bank were resuscitated under suitable conditions and cultivated in NNN and RPMI-1640 media. Then, the isolates were allowed to enter the logarithmic phase in a 26ºC incubator and DNA isolations were made using the "High Pure PCR Template Preparation Kit". Real-time polymerase chain reaction (Rt-PCR) melting analyzes were applied to the DNAs obtained by using primers and probes specific to the internal transcribed spacer-1 (ITS-1) gene region of Leishmania. After RNA isolation from promastigote suspension, cDNA synthesis was performed by reverse transcription. After gel electrophoresis with PCR amplification products, dsRNA band formation was evaluated in terms of LRV2 positivity under ultraviolet light. Among the 20 examined Leishmania spp. isolates (10 L.tropica and 10 L.major), four (three L.tropica, one L.major) were found to be positive for the presence of LRV2. Although the mechanism of LRV in recent studies has not been fully understood, it is known that it exacerbates the clinic of the disease and even has an effect on the formation of drug resistance by the parasite. It is important to obtain data on the presence of LRV in our country and to contribute to various clinical, drug development, prevalence studies, diagnosis and treatment of the disease in the future.

Gender Determination by PCR Assay for The Sex-Determining Region Y(SRY) Gene Amplification in Linnaeus’s Two-Toed Sloth (Choloepus Didactylus)

In Linnaeus’s two-toed sloths (Choloepus didactylus), there is no distinct sexual dimorphism. It is an obstacle for gender determination from the external genitalia, especially in newborns or young sloths. Hence, easy, rapid, and reliable genetics-based methods for gender identification of the sloths are needed to continue captive breeding more successfully. In this study, a PCR-based technique that allows gender determination of two-toed sloths by using a sex-determining region Y (SRY) gene marker was described. The hair samples from young (suspect gender) and adult sloths (known gender) were used in genetic analysis. Initially, genomic DNA was isolated from hair root samples using Roche high pure PCR template preparation kit. The SRY primers were specifically designed based on the NCBI and Ensembl databases, and they were verified with the BLAST program concerning the two-toed sloth genome. PCR amplification with the SRY-specific primers was carried out by a programmable thermal cycler device using FastStart High Fidelity PCR System, Roche dNTPack. The samples were then electrophoresed on 2% agarose gels and were visualized by a gel documentation and analysis system. A specific band in the electrophoresis pattern is diagnostic for a male individual with a partial SRY region. Hence, the analysis demonstrated that the samples belonged to a male two-toed sloth. Two-toed sloth species are commonly preferred animals in zoos. Gender determination is inevitable for these animals in captivity to be raised successfully and healthily. Molecular genetic techniques allow high efficiency in taxonomic evaluations and gender identification in species that do not display sexual dimorphism. The PCR assay described here may be helpful for a rapid genetic analysis that can be widely used in gender determination for two-toed sloths.

MDR1 Geni ile Ankilozan Spondilit Arasındaki İlişkinin İncelenmesi: Olgu Kontrol Araştırması

Objective: In this study, the relationship between the MDR1 gene and ankylosing spondylitis (AS) was investigated. Genetic and environmental factors are known to play an important role in the pathogenesis of AS. It is obvious that different patients have different reactions to prescribed drugs and drug dosages during standard treatment. One of these factors is the P-glycoprotein encoded by the MDR1 gene. The most widely studied alleles of this gene are rs1045642-NM_000927.4:c.3435T>C, p. Ile1145Ile (C3435T), rs2032582-NM_000927.4:c.2677T>G/A, p. Ser893Ala/Thr (G2677T/A) and silent rs1128503-NM_000927.4:c.1236T>C, p. Gly412Gly (C1236T). Material and Methods: The study group was formed from 34 patients with biological therapy, 32 patients with other treatments (66 AS patients in total) and 32 healthy individuals. DNA isolation was performed using a High Pure PCR Template Preparation Kit. RNA isolation was performed with TRIzol manual isolation. For quantitative real-time polymerase chain reaction, a Roche LightCycler FastStart DNA Master device HybProbe was used. Results: There was no statistically significant difference between groups regarding the investigated polymorphisms. In the biological therapy [tumor necrosis factor alpha (TNF-α) inhibitor] group, a significant decrease was found in MDR1 gene expression (p<0.001). Conclusion: In conclusion, we could say that there is no relation between single-nucleotide polymorphism and treatment choice and response for AS patients. Decreased MDR1 expression can be explained by the possible downregulatory effect of TNF-α inhibitors.

The influence of RS738409 I148M polymorphism of patatin-like phospholipase domain containing 3 gene on the susceptibility of non-alcoholic fatty liver disease

We aimed to elucidate the frequency of polymorphic genotypes and alleles of patatin-like phospholipase domain containing 3 rs738409 polymorphism and its possible associations with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis in a cohort from Turkey.We enrolled 200 patients diagnosed with NAFLD and genotyped for rs738409 I148M polymorphism by real-time polymerase chain reaction, particularly by melting curve analysis. SPSS analysis software was used for statistical significance. Continuous variable values were expressed as mean ± standard deviation. Significant statistical level was chosen as p = 0.05.Our results demonstrate in a cohort from Turkey that rs738409 C > G polymorphism (I148M) of patatin-like phospholipase domain containing 3 gene is significantly able to affect individuals to have NAFLD in unadjusted regression model.Consistent with the previous studies in other populations, our study group showed a significantly higher risk of having NAFLD in unadjusted regression model but not in the adjusted model indicating that non-genetic factors such as age and sex may be responsible for the association. However, independent studies need to validate our findings with a larger group of NAFLD patients, as well as in different ethnic cohorts.

Frequency of IKZF1 Deletions in a Peruvian Population with B-Cell Acute Lymphoblastic Leukemia

Frequency of IKZF1 Deletions in a Peruvian Population with B-cell Acute Lymphoblastic Leukemia Background: B-cell Acute Lymphoblastic Leukemia (B-ALL) is an aggressive disease with worse outcomes in older patients, and latino ethnicity. Additionally, Latino populations are at higher risk of developing B-ALL.IKZF1is an essential lymphoid transcription factor with deletions (ΔIKZF1)implicated in treatment failure and relapses. We aimed to evaluate the frequency ofIKZF1deletions in a cohort of Peruvian patients with newly diagnosed B-ALL. Methods: We collected diagnostic bone marrow samples from 41 consecutive patients with B-ALL diagnosed between 2015-2019 at Instituto Nacional de Enfermedades Neoplasicas (INEN; Lima, Peru). Bone marrow samples were cryopreserved prior to induction treatment. DNA was extracted using High Pure PCR Template Preparation Kit (Roche) at INEN. Samples with adequate DNA were screened forΔIKZF1by multiplex endpoint PCR covering four main deletions - dominant negative Δ4-7 or the loss of function Δ2-7, Δ4-8, and Δ2-8 IKZF1 deletions at UCL Cancer Insitute (London, UK) using the primers described by Caye et. al. We analyzed outcomes byIKZF1status. Results: Forty-one cases were enrolled during the study period. Clinical characteristics are presented in Table 1. Median age was 20 years[1-63]. Fifteen∆IKZF1cases (37%) were detected (67%BCR-ABL1 negand 33%BCR-ABL1pos).Cases withΔIKZF1were older than those with wild-typeIKZF1(median age 31 vs 13 years, p=0.002). Median presenting white blood count (WBC) was 48 x109/L [R:2-218], with a higher WBC inΔIKZF1compared to wild-type (87 vs 24 x109/L, p=0.001). The most frequent deletion was ∆4-7 (sevenBCR-ABL1 negand threeBCR-ABL1 pos) additional deletions are described in table 2. All patients received intensive 'pediatric-based' treatment, 21 with BFM-2009 and 19 with the CALGB 10403 protocol. CR rates after induction were 67% and 92% for∆IKZF1and wild-type cases, respectively. Eleven (73%) of patients with∆IKZF1subjects (73%) were MRD positive by flow cytometry after induction compared to 11 (44%) among wild-type. At a median follow-up of 2 years EFS was 38% in the∆IKZF1group and 58% in the wild type group, correspond OS was 38% and 58%, respectively. Conclusion: A high frequency of IKZF1 deletions was found in a Peruvian population with B-ALL and was associated with older age and higher presenting white blood counts. Prospective studies with larger Latino population are warranted to confirm this finding. Disclosures No relevant conflicts of interest to declare.

The Healthy Airway Mycobiome in Individuals of Asian Descent

The Healthy Airway Mycobiome in Individuals of Asian Descent

Identifying Variable Number of Tandem Repeat Alleles in Phenylalanine Hydroxylase Gene in Patients with Phenylketonuria in Golestan Province, Iran

Background and Objective: Phenylketonuria (PKU) is a metabolic disorder that is caused by mutations in the phenylalanine hydroxylase (PAH) gene. The multiplicity of mutations in the PAH gene of PKU leads to the cases, in which the pathogenic mutation cannot be detected. In these cases, the variable number of tandem repeat (VNTR), which is the polymorphic marker associated with the PAH gene, is used to identify PKU carriers. The present study was conducted to investigate the allele frequency of this marker in PKU population of Golestan Province (north of Iran). Materials and Methods: In this cross-sectional descriptive study, 26 non-relative PKU patients (1-23 years old, phenylalanine: 4.5-250 mg/dl) were identified from different regions of Golestan Province within a one-year period (2016). Genomic DNA was then extracted from leukocytes using the high pure PCR template preparation kit (Roche) and the fragments containing the PAH VNTR were evaluated using PCR-sequencing method. Results: PCR products of PAH VNTR alleles produced 334, 454, 484, 514, and 604 bp fragments. They were corresponding to the presence of alleles with 3, 7, 8, 9, and 12 copies of the repeat units, respectively. In addition, the distribution of VNTR alleles was as follows: 28.85, 28.85, 17.3, 19.23, and 5.77. Conclusion: This study is the first report regarding the genetic structure of PKU population using PAH VNTR alleles in Golestan Province. Considering the population diversity in Iran, it is necessary to investigate the frequency and distribution of VNTR alleles in different parts of the country.

The Quality of DNA Isolated from Processed Food and Feed via Different Extraction Procedures.

The extraction of DNA is a critical step for species identification by PCR analysis in processed food and feed products. In this study, eight DNA extraction procedures were compared—DNeasy Blood and Tissue Kit, DNeasy mericon Food Kit, chemagic DNA Tissue 10 Kit, Food DNA Isolation Kit, UltraPrep Genomic DNA Food Mini Prep Kit, High Pure PCR Template Preparation Kit, phenol—chloroform extraction, and NucleoSpin Food—Using self-prepared samples from both raw and heat-processed and/or mechanically treated muscles and different types of meat products and pet food (pork, beef, and chicken). The yield, purity, and suitability of DNA for PCR amplification was evaluated. Additionally, comparisons between the effectiveness of various extraction methods were made with regard to price, and labor- and time-intensiveness. It was found that the DNeasy mericon Food Kit was the optimal choice for the extraction of DNA from raw muscle, heat-treated muscle, and homemade meat products from multiple and single species.

Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays.

BackgroundPolymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process.Methodology/Principal findingsWe assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately.Conclusions/SignificanceThis is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers.

Optimasi Suhu Annealing Tiga Regio Berbeda Isolat Multidrug Resistance Mycobacterium Tuberculosis dengan Metode Multiplex Polymerase Chain Reaction (ANNEALING TEMPERATURE OPTIMIZATION ON THREE DIFFERENT REGIONS OF MYCOBACTERIUM TUBERCULOSIS MULTIDRUG RESISTANCE ISOLATE USING MULTIPLEX POLYMERASE CHAIN REACTION)

Multiplex PCR is a method used to amplify more than one target sequences simultaneously. The aimof this research was to optimize PCR on the region of inhA promoter, inhA gene and katG gene usingMultidrug Resistance Tuberculosis (MDR-TB) Isolate. Isolation of DNA was done by using High Pure PCRTemplate Preparation Kit. Amplification process was done by Multiplex PCR usingthree pairs primers i.e.mabA-inhA-promoter-FS and mabA-inhA-promoter-R,inhA (F) and inhA (R) and KG24F and KG60R.Amplification process started by predenaturation at 95°C for 15 minutes, followed by 45 cycles consistingof denaturation at 94°C for 1 minute, annealing at 56°C, 57°C and 58°C for 1 minute and 20 seconds andextension at 72°C for 2 minutes. Then it is finished by postextension at 72°C for 10 minutes. PCR productwas detected by electrophoresis and visualized under UV Transiluminator. Annealing temperature of56°C resulted in a thicker, clearerandaccording to the desired size as compared to that of with 57°C and58°C. Conclusion that obtained was annealing temperature 56°C was optimum annealing temperatureon inhA promoter, inhA gene and katG gene region of Mycobacterium tuberculosis Multidrug Resistanceisolate using Multiplex Polymerase Chain Reaction.

Association of KCNJ11 (E23K) gene polymorphism with susceptibility to type 2 diabetes in Iranian patients.

Background:Type 2 diabetes (T2D) is a multifactorial disease with susceptibility of several genes that are related to T2D. Insulin secretion pathway starts with potassium channels in pancreatic beta cells. KCNJ11 gene encodes ATP-sensitive potassium channel subunits. Some studies suggested that KCNJ11 (E23K) mutation increases the risk of T2D. Therefore, present study was designed to investigate the association between E23K polymorphism of KCNJ11 gene and type 2 diabetes mellitus (T2DM) in the Iranian population.Materials and Methods:The type of study was case-control and 40 unrelated subjects, including 20 healthy controls and 20 diabetic patients were recruited (diagnosed based on American Diabetes Association criteria). Blood samples were used for isolation of genomic deoxyribonucleic acid (DNA). Having extracted the genomic DNA from human blood leukocytes by means of High Pure PCR Template Preparation Kit, PCR-restriction fragment length polymorphism method was used to detect KCNJ11 E23K gene polymorphism. BanII restriction enzyme was used for digestion. Data were analyzed using Chi-square or Fisher exact test or independent t-test, as appropriate. P < 0.05 was considered.Results:We found that the carrier homozygous for KK genotype are susceptible to T2D (0.049) and in patients the frequency of K allele was higher than control subjects (0.048).Conclusion:The present study suggests that KCNJ11 (E23K) gene polymorphism is associated with T2DM.

Distinct Associations Between UGT1A1 Gene Promoter Polymorphism and Hyperbilirubinemia in Korean CML Patients Treated with Nilotinib and Radotinib

Background: Second-generation BCR-ABL tyrosine kinase inhibitors (2nd generation TKIs) including nilotinib (NIL) and radotinib (RAD) are approved for the treatment of newly diagnosed and other TKI failed chronic myeloid leukemia (CML). Uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) gene promoter polymorphism, distinct number of TA repeats in promoter, has been reported for an association with hyperbilirubinemia on nilotinib therapy (Singer et al. Leukemia (2007) 21, 2311–2315). However, the distribution of UGT1A1 (TA)7 repeat polymorphism differs greatly between Caucasians and Japanese, namely, the frequency of UGT1A1(TA)7 repeat polymorphism is high in Caucasians, whereas it is low in Asians (Beutler et al. Proc. Natl. Acad. Sci. USA 95 (1998) 95, 8170–8174 ).Aims: The aim of this study was to investigate the association between UGT1A1 gene promoter polymorphism and hyperbilirubinemia in Korean CML patients treated with NIL and RAD as a frontline therapy.Methods: A total of 88 newly diagnosed chronic phase CML patients who treated with frontline NIL (n=39) and RAD (n=49) was screened for UGT1A1 promoter polymorphism genotype analysis. We used the High Pure PCR Template Preparation Kit (Roche, Germany) to prepare genomic DNA from whole blood and genotyped by direct sequencing of the 253- to 255-bp fragments produced by PCR.Results: The (TA)6/(TA)6 homozygote and (TA)6/(TA)7 heterozygote were seen in all genotyped population and frequency of (TA)6/(TA)6 homozygote was 79.5% (70/88) in our patients. (TA)6/(TA)6 homozygote predominated with 81.6% of the alleles in the RAD group and 76.9% in the NIL group. Relative riskfor each genotype presented in Table 1, with hyperbilirubinemia defined as CTCAE grade 2 or greater observed at any post-baseline point. The relative risks for 6/6 genotype versus 6/7 genotype was 3.5 with 95% CIs of (0.6, 18.9) in the RAD group compared with 8.1 (1.5, 43.8) in the NIL group and NIL group showed significantly high association with UGT1A1 gene promoter polymorphism (P<0.05).Conclusions: This finding suggests that UGT1A1 gene promoter polymorphism may be an important determinant of hyperbilirubinemia in CML patients with NIL therapy, but not in RAD. However other mechanisms should be explored in patients who have significant hyperbilirubinemia with RAD therapy. Updated data with longer follow-up duration will be presented in the meeting.Table 1Relative risk calculations for prevalence of hyperbilirubinemiaRadotinibNilotinibMax grade=,<1Max grade>,=2TotalMax grade=,<1Max grade>,=2Total(TA)6/(TA)6202040(TA)6/(TA)62643050.00%50.00%65.00%10.00%(TA)6/(TA)7279(TA)6/(TA)745922.22%77.78%44.44%55.56%Total222749Total30939Relative risk=3.5, 95% CI of (0.6, 18.9)Relative risk=8.1, 95% CI of (1.5, 43.8)Abbreviation: CI, confidence interval. DisclosuresNo relevant conflicts of interest to declare.

Investigation of sensitivity, specificity and accuracy of Tetra primer ARMS PCR method in comparison with conventional ARMS PCR, based on sequencing technique outcomes in IVS-II-I genotyping of beta thalassemia patients

PurposeBeta thalassemia is one of the most important hematic diseases all around the world and solving the problems caused by this abnormality is strongly dependent on precise detection and reliable screening of high-risk couples. The aim of our study was the investigation of sensitivity, specificity and accuracy of Tetra primer ARMS PCR method comparing with conventional ARMS PCR, based on sequencing technique outcomes for genotyping of IVS-II-I mutation in beta thalassemia patients. MethodsFifty seven samples including two homozygote, 49 heterozygote and 6 normal specimens were analyzed by Tetra primer ARMS PCR and conventional ARMS PCR methods. DNA was extracted by the standard method of salting out for leukocyte genomic DNA extraction of blood specimens and a high pure PCR template preparation kit was used for DNA purification of CVS samples. The results obtained by Tetra primer ARMS PCR and conventional ARMS PCR methods were compared with gold standard technique, i.e. sequencing. ResultsAll three parameters including specificity, sensitivity and accuracy were 100% for Tetra primer ARMS PCR method, while they were 100%, 92.45% and 92.7% for conventional ARMS PCR technique respectively. Comparing with Tetra primer ARMS PCR which represented 100% agreement with sequencing method, conventional ARMS PCR technique only showed 47.1% agreement, because of 4 discordant results. ConclusionTetra primer ARMS PCR method is an almost reliable, sensitive and accurate technique and it is suggested that it can be used as a complementary method for diagnostic cases instead of conventional ARMS PCR method. This suggestion originated with perfect rate of agreement between outcomes of sequencing method, as a gold standard method of detecting the mutations, and Tetra primer ARMS PCR technique comparing with conventional ARMS PCR method.

DNA extraction from long-term stored urine

BackgroundTraditionally, for DNA analyses, DNA is recovered from buffy coats. Since DNA in urine has been reported to deteriorate quickly, this option is often not considered. To complete our DNA database in patients with ANCA-associated vasculitis, we aimed to extract DNA from stored urine.MethodsUrine was stored at the time of kidney biopsy from patients included in our regional kidney biopsy database, who had given informed consent for further study. Urine was subsequently filtered, dialyzed, concentrated and freeze dried and finallyresolubilized and centrifuged. DNA was extracted using the high pure PCR template preparation kit (Roche Diagnostics). Next, concentration and purity were determined by Nanodrop analysis and by Quant-iT analysis.ResultsOne hundred and eighty-one patients with ANCA-associated vasculitis were included. Of 114 patients (63%), DNA was available. From 53 of the remaining 67 patients, stored urine was available. Of the 53 samples that were processed, 46 (86.8%) yielded DNA with a mean concentration of 258.7 ng/μL (range 33.2-529) with a mean purity ratio of 1.81 (λ 260/280).ConclusionDNA extraction from fresh urine has been described before, yielding DNA usable for PCR analysis in healthy subjects. Storage of fresh urine at 4°C or lower temperatures results in significant degradation of the DNA, making recovery of DNA more difficult with longer periods of storage. In the current study, we demonstrated that DNA could be retrieved from subsequently filtered, dialyzed, concentrated and freeze dried urine that was stored at room temperature. In addition, we demonstrated tthat this DNA could be used for PCR analysis. This method is useful when no other material from these patients is available.

Chronic hepatitis B and IL28B rs12979860 polymorphism: preliminary study

The aim of this study is to evaluate the role of IL28B rs12979860 polymorphism on pegylated interferon (peg IFN) and oral antiviral treatment in chronic hepatitis B (CHB) patients and to investigate the relationship between the severities of illness with this polymorphism. 74 CHB patients who are received treatment, 61 asymptomatic carriers and 40 healthy controls were recruited in this study. Genomic DNA of controls and patients were extracted from whole blood using High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany) according to the manufacturer's instructions and stored at 4 °C. Genotype distribution of the IL-28B polymorphism at position -3176C/T (rs12979860) (LightMix Kit IL28B, Cat.-No. 40-0588-32 TIB MOLBIOL, Berlin, Germany) was detected by real time PCR (Roche Diagnostics, Manheim, Germany). Thirty of the patients with CHB received peg IFN-α treatment. There were no significant difference between groups by means of age, gender and IL28B rs12979860 polymorphism (p = 0.122, p = 0.07, p = 0.376 respectively). Patients with chronic hepatitis were categorized as grade and stage (minimal, moderate and severe) and then were analyzed for the polymorphism. There was no effect of IL28B -3176 C/T polymorphism on severity of illness (p = 0.293 for grade, p = 0.911 for stage). When the CHB treatment monitored in different time arrivals (beginning, 3th, 6th and 12th months of the treatment) in order to see if there was an effect on virological and biological response none of the genotypes of IL28B -3176C/T polymorphism altered peg IFN or oral antiviral treatment process. There are conflicting results about the role of IL28B rs12979860 polymorphism in CHB in the literature. In this preliminary study, we observed that IL28B rs12979860 polymorphism was not related with severity of illness and also was not effective on treatment response.

Comparison of DNA Extraction Kits for Detection of Burkholderia pseudomallei in Spiked Human Whole Blood Using Real-Time PCR

Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1) assay. Crossing threshold (CT) values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×104 colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen.

217 TRANSCRIPT ABUNDANCE OF CATHEPSIN GENES IN CUMULUS CELLS AS A MARKER OF CATTLE OOCYTE QUALITY

Dynamics of follicular growth and atresia is closely connected with apoptosis. Cathepsins (CTS) are involved in diverse biological functions, whereas one member of this family, cathepsin B, plays a major regulatory role in the process of apoptosis. Oocyte quality is a complex trait shaped by the follicular components (e.g. cumulus cells, CC; follicular fluid, FF). A negative relationship between relative transcript abundance (RA) of CTSB, CTSS, and CTSZ genes in CC with the quality of corresponding oocytes was reported in cattle (Bettegowda et al. 2008). Fatty acid (FA) composition of the FF and mtDNA copy number in the oocyte are other markers of oocyte quality. Therefore, in this study, we analysed relations between selected traits of the 3 follicular components (FF, CC, oocyte) within the individual follicle with the focus on oocyte quality. The experiment was based on cumulus–oocyte-complexes (COC) and FF obtained from individual follicles of slaughterhouse ovaries. Each follicle was measured and assigned into 1 of 3 classes (small &lt;6 mm; medium 6 to 8 mm; large &gt;8 mm). The COC morphology (grades 1 to 4) was evaluated according to Stojkovic et al. (2001). The following analyses were performed: CC, mRNA abundance of CTSB, CTSS, CTSZ, and CTSK genes (real-time PCR, 100 replicates, ACTB as a reference gene); the oocyte, mtDNA copy number (real-time PCR, 93 replicates, COX1 gene); and FF and FA composition (gas chromatography). The following procedures were employed: total RNA isolation, mirVana Paris Kit (Ambion); total DNA isolation, High Pure PCR Template Preparation Kit (Roche, Indianapolis, IN, USA); cDNA synthesis, Transcriptor High Fidelity cDNA Synthesis Kit (Roche); and the standard curve method, to analyse the qPCR data. For statistical analysis, the Kruskal-Wallis test as well as Spearman rank correlation were applied. The highest RA of CTSB gene was noted in CC from the grade 3 COC (P &lt; 0.05), whereas that of CTSK and CTSZ genes in CC from the grade 4 COC (P &lt; 0.01). Because grade 3 and 4 COC are not suitable for IVM, we assumed that high RA of CTS gene in CC may indicate reduced quality of the corresponding oocyte. Surprisingly, the highest RA for CTSB gene was observed in CC from the medium follicles (P &lt; 0.05). Significant (P &lt; 0.05) correlations were estimated between the following: RA of CTSB gene in CC and mtDNA copy number in the oocyte (r = 0.27), RA of CTSB gene in CC and C18.3 n-3 concentration in FF (r = 0.32), RA of CTSZ gene in CC and C18.3 n-3 concentration in FF (r = 0.37), as well as RA of CTSZ gene in CC and n-3 concentration in FF (r = 0.34). Although an increase in RA of the CTS genes in CC was accompanied by the inferior oocyte morphology, it was also correlated with higher mtDNA copy number in the oocyte and FA content in FF. The last 2 features have been previously attributed to oocytes of better quality, what contrasts with the high RA of the CTS genes. Thus, higher RA of CTS genes within CC may not mark the bovine oocyte of reduced quality. Funding–National Science Center, grant no. N N302 604438.