Identification of potential cancer targets from diagnostic and prognostic gene signatures

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 20096-20096
Author(s):  
G. Vansant ◽  
K. Oades ◽  
M. Pickering ◽  
J. Monforte
Keyword(s):  
Reverse Transcription ◽  
Metastatic Potential ◽  
Universal Primers ◽  
Pcr Analysis ◽  
Mucin 1 ◽  
Universal Primer ◽  
Specific Sequence ◽  
Gene Signatures ◽  
Prognostic Gene ◽  
Pcr Products

20096 Background: The purpose of this study was to determine the potential of gene signature development to also identify leads among different tumor types compared to normal cells. The theory is that cancer cells will express unique markers that are related to their growth and metastatic potential and therefore be potential targets for therapeutics. Methods: Multiplex PCR Approximately 100 genes were chosen to produce 3 multiplexes for RT-PCR analysis. These genes were identified from gene signatures found in peer reviewed manuscripts. RT and PCR primer pairs designed for each gene to be monitored are chimeric, with a gene specific sequence and a universal sequence common to all forward and reverse primers, respectively. A pair of universal primers that recognize the universal sequences in the chimeric primers are included in the reaction in great excess, with one fluorescently labeled. After reverse transcription and a few rounds of PCR, these universal primers drive the reactions, so all the PCR products are essentially amplified by the universal primer set. The chimeric primers are designed to produce PCR products that all have a difference in length of approximately 5 base pairs, resulting in a stratified set of labeled PCR products. 25ng of total RNA from each tumor derived cell line was used as template for the reverse transcription reactions. Half of these reactions were carried over as templates for the PCR reactions. The PCR products were fluorescently labeled during the PCR reaction and analyzed on a capillary electrophoresis system. Results: Many genes were expressed at high levels across all cancer cell types compared to control cells including an A kinase anchoring protein, cyclin-E2, JPO1, mucin 1, and the transferrin receptor CD 71. Conclusions: Analysis of the expression of genes that are part of diagnostic and prognostic gene signatures in tumor related cell lines revealed many genes whose expression were altered in tumor derived cells that are potentially related to the ability of the carcinogenic cells to proliferate and metastasize. Subsequently, a secondary benefit of the development of prognostic or diagnostic gene signatures with statistical analysis, may be the elucidation of potential therapeutic targets for the treatment of cancer. No significant financial relationships to disclose.

scholarly journals Determination of m6A frequency utilizing 4SedTTP-RT Ligation Assisted PCR (SLAP) in viral and cellular long non-coding RNAs

2021 ◽  
Author(s):  
Sarah Elizabeth Martin ◽  
Huachen Gan ◽  
Joanna Sztuba-Solinska
Keyword(s):  
Reverse Transcription ◽  
Quantitative Assessment ◽  
Biological Significance ◽  
Pcr Analysis ◽  
Rna Biology ◽  
Pcr Products ◽  
Non Coding Rnas ◽  
Nucleotide Resolution ◽  
Target Rna

N6-methyladenosine is one of the most abundant epitranscriptomic signatures that can affect every aspect of RNA biology, from structure and stability to intra- and intermolecular interactions. The accurate quantitative assessment of RNA stoichiometry at single-nucleotide resolution is a prerequisite to evaluate the biological significance of m6A in the context of specific RNA. We have developed a new method, termed 4-Selenothymidine 5′-triphosphate reverse transcription and Ligation Assisted PCR analysis (SLAP), for quantitative and unbiased assessment of the m6A fraction on target RNA. The inclusion of thymidine triphosphate derivative during reverse transcription discourages base pair formation with m6A resulting in the reaction's cessation, while maintaining normal A-T base pairing. The site-specific ligation of the resulting cDNAs with adapters, followed by amplification, generates two distinct products that reflect the modified and unmodified fraction of the analyzed RNA. These PCR products are subsequently separated by gel electrophoresis and quantified using densitometric analysis. We applied the SLAP to verify the position and assess the frequency of m6A sites present on two exemplary long non-coding RNAs. We assessed the SLAP specificity, accuracy, and sensitivity, proving the applicability of this method for the m6A analysis on less abundant transcripts. Overall, this method constitutes an extension of the bird's-eye view of RNA m6A landscape provided by epitranscriptome-wide analyses by delivering quantitative assessment of modification frequency and can therefore aid the understanding of the consequences of m6A on biological processes.

scholarly journals Reverse Transcription-PCR Analysis of Bottled and Natural Mineral Waters for the Presence of Noroviruses

2003 ◽  
Vol 69 (11) ◽  
pp. 6541-6549 ◽  
Author(s):  
Gilbert Thierry Lamothe ◽  
Thierry Putallaz ◽  
Han Joosten ◽  
Joey D. Marugg
Keyword(s):  
Reverse Transcription ◽  
Membrane Filtration ◽  
Limit Of Detection ◽  
Mineral Waters ◽  
Pcr Analysis ◽  
Rna Sequences ◽  
Natural Mineral ◽  
Reverse Transcription Pcr ◽  
Viral Particles ◽  
Nonspecific Amplification

ABSTRACT A seminested reverse transcription-PCR method coupled to membrane filtration was optimized to investigate the presence of norovirus (NV) RNA sequences in bottled and natural mineral waters. The recovery of viral particles by filtration varied between 28 and 45%, while the limit of detection of the overall method ranged from 6 to 95 viral particles. The assay was broadly reactive, as shown by the successful detection of 27 different viral strains representing 12 common genotypes of NVs. A total of 718 bottled and natural mineral water samples were investigated, including 640 samples of finished, spring, and line products (mostly 1 to 1.5 liters), collected from 36 different water brands of various types and from diverse geographic origins over a 2-year period. In addition, 78 samples of larger volume (10 and 400 to 500 liters) and environmental swabs were investigated. From the 1,436 analyses that were performed for the detection of NVs belonging to genogroups I and II, 34 samples (2.44%) were presumptively positive by seminested RT-PCR. However, confirmation by DNA sequence analysis revealed that all presumptive positive results were either due to nonspecific amplification or to cross-contamination. In conclusion, these results do not provide any evidence for the presence of NV genome sequences in bottled waters.

scholarly journals Selection of Valid Reference Genes for Reverse Transcription Quantitative PCR Analysis inHeliconius numata(Lepidoptera: Nymphalidae)

2016 ◽  
Vol 16 (1) ◽  
pp. 50 ◽  
Author(s):  
Florence Piron Prunier ◽  
Mathieu Chouteau ◽  
Annabel Whibley ◽  
Mathieu Joron ◽  
Violaine Llaurens
Keyword(s):  
Quantitative Pcr ◽  
Reverse Transcription ◽  
Reference Genes ◽  
Pcr Analysis ◽  
Reverse Transcription Quantitative Pcr ◽  
Selection Of

scholarly journals Rapid Detection of Phytophthora infestans in Late Blight-Infected Potato and Tomato Using PCR

Plant Disease ◽  
1997 ◽  
Vol 81 (9) ◽  
pp. 1042-1048 ◽  
Author(s):  
C. L. Trout ◽  
J. B. Ristaino ◽  
M. Madritch ◽  
T. Wangsomboondee
Keyword(s):  
Late Blight ◽  
Phytophthora Infestans ◽  
Pcr Amplification ◽  
Accurate Method ◽  
Phytophthora Species ◽  
Universal Primers ◽  
Direct Pcr ◽  
Field Samples ◽  
Pcr Products ◽  
Oomycete Pathogen

Late blight caused by the oomycete pathogen Phytophthora infestans is a devastating disease of potato and tomato worldwide. A rapid and accurate method for specific detection of P. infestans is necessary for determination of late blight in infected fruit, leaves, and tubers. Ribosomal DNA (rDNA) from four isolates of P. infestans representing the four genotypes US1, US6, US7, and US8 was amplified using polymerase chain reaction (PCR) and the universal primers internal transcribed spacer (ITS) 4 and ITS5. PCR products were sequenced using an automated sequencer. Sequences were aligned with published sequences from 5 other Phytophthora species, and a region specific to P. infestans was used to construct a PCR primer (PINF). Over 140 isolates representing 14 species of Phytophthora and at least 13 other genera of fungi and bacteria were used to screen the PINF primer. PCR amplification with primers PINF and ITS5 results in amplification of an approximately 600 base pair product with only isolates of P. infestans from potato and tomato, as well as isolates of P. mirabilis and P. cactorum. P. mirabilis and P. cactorum are not pathogens of potato; however, P. cactorum is a pathogen of tomato. P. infestans and P. cactorum were differentiated by restriction digests of the amplified product. The PINF primer was used with a rapid NaOH lysis technique for direct PCR of P. infestans from infected tomato and potato field samples. The PINF primer will provide a valuable tool for detection of P. infestans in potatoes and tomatoes.

scholarly journals First Report of a Group 16SrII Phytoplasma Infecting Chickpea in Oman

Plant Disease ◽  
2006 ◽  
Vol 90 (7) ◽  
pp. 973-973 ◽  
Author(s):  
N. A. Al-Saady ◽  
A. M. Al-Subhi ◽  
A. Al-Nabhani ◽  
A. J. Khan
Keyword(s):  
Nested Pcr ◽  
Animal Feed ◽  
Restriction Enzymes ◽  
Universal Primers ◽  
Polymorphism Analysis ◽  
Disease Symptoms ◽  
First Report ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Group 2

Chickpea (Cicer arietinum), locally known as “Dungo”, is grown for legume and animal feed mainly in the interior region of Oman. During February 2006, survey samples of chickpea leaves from plants showing yellows disease symptoms that included phyllody and little leaf were collected from the Nizwa Region (175 km south of Muscat). Total nucleic acid was extracted from asymptomatic and symptomatic chickpea leaves using a cetyltrimethylammoniumbromide method with modifications (3). All leaf samples from eight symptomatic plants consistently tested positive using a polymerase chain reaction assay (PCR) with phytoplasma universal primers (P1/P7) that amplify a 1.8-kb phytoplasma rDNA product and followed by nested PCR with R16F2n/R16R2 primers yielding a product of 1.2 kb (2). No PCR products were evident when DNA extracted from healthy plants was used as template. Restriction fragment length polymorphism analysis of nested PCR products by separate digestion with Tru9I, HaeIII, HpaII, AluI, TaqI, HhaI, and RsaI restriction enzymes revealed that a phytoplasma belonging to group 16SrII peanut witches'-broom group (2) was associated with chickpea phyllody and little leaf disease in Oman. Restriction profiles of chickpea phytoplasma were identical with those of alfalfa witches'-broom phytoplasma, a known subgroup 16SrII-B strain (3). To our knowledge, this is the first report of phytoplasma infecting chickpea crops in Oman. References: (1) A. J. Khan et al. Phytopathology, 92:1038, 2002. (2). I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998 (3) M. A. Saghai-Maroof et al. Proc. Natl. Acad. Sci. USA. 81:8014, 1984.

scholarly journals Specific Detection of Fasciola hepatica and F. gigantica in Infected Domesticated Animals Using High-Resolution Melting Analysis (HRM)

2020 ◽  
Author(s):  
Zeinab MOGHADAMIZAD ◽  
Ahmad HOSSEINI-SAFA ◽  
Mehdi MOHEBALI ◽  
Peyman HEYDARIAN ◽  
Mojgan ARYAEIPOUR ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Fasciola Hepatica ◽  
High Resolution Melting Analysis ◽  
Universal Primers ◽  
Specific Primer ◽  
Universal Primer ◽  
Melting Analysis ◽  
Liver Flukes ◽  
Fasciola Spp

Background: It is difficult to make an exact morphological distinction between Fasciola hepatica and Fasciola gigantica. We used High Resolution Melting analysis (HRM) method to differentiate the F. hepatica species from F. gigantica in order to differentiate them. Methods: Overall, 80 adult liver flukes were collected from infected slaughtered animals including cattle, sheep and goats from Lorestan Province, western Iran from Sep 2015 to Aug 2017. Genomic DNA was extracted using commercial DNA extraction kit. The multilocus sequences of mDNA including COX1, COX3 and ND6 were amplified employing real-time PCR & HRM analysis. Specific and universal primer pairs were designed for differentiation Fasciola spp. Results: Universal primers cannot be used to distinguish between these two species, but in the contrary, specific primer pairs of each species could differentiate them properly. Molecular identification using specific primer pairs were consistent. Conclusion: HRM is a simple, fast and reliable method for detecting and differentiating F. hepatica from F. gigantica and can be used for diagnostic and epidemiological purposes.

scholarly journals First report of Coleus blumei viroid 1 in commercial Coleus blumei in the United States

Plant Disease ◽  
2021 ◽  
Author(s):  
Gardenia Orellana ◽  
Alexander V Karasev
Keyword(s):  
United States ◽  
Leaf Tissue ◽  
The United States ◽  
Universal Primers ◽  
Rt Pcr ◽  
Universal Primer ◽  
Tissue Samples ◽  
First Report ◽  
Specific Pcr ◽  
Coleus Blumei

Coleus scutellarioides (syn. Coleus blumei) is a widely grown evergreen ornamental plant valued for its highly decorative variegated leaves. Six viroids, named Coleus blumei viroid 1 to 6 (CbVd-1 to -6) have been identified in coleus plants in many countries of the world (Nie and Singh 2017), including Canada (Smith et al. 2018). However there have been no reports of Coleus blumei viroids occurring in the U.S.A. (Nie and Singh 2017). In April 2021, leaf tissue samples from 27 cultivars of C. blumei, one plant of each, were submitted to the University of Idaho laboratory from a commercial nursery located in Oregon to screen for the presence of viroids. The sampled plants were selected randomly and no symptoms were apparent in any of the samples. Total nucleic acids were extracted from each sample (Dellaporta et al. 1983) and used in reverse-transcription (RT)-PCR tests (Jiang et al. 2011) for the CbVd-1 and CbVd-5 with the universal primer pair CbVds-P1/P2, which amplifies the complete genome of all members in the genus Coleviroid (Jiang et al. 2011), and two additional primer pairs, CbVd1-F1/R1 and CbVd5-F1/R1, specific for CbVd-1 and CbVd-5, respectively (Smith et al. 2018). Five C. blumei plants (cvs Fire Mountain, Lovebird, Smokey Rose, Marrakesh, and Nutmeg) were positive for a coleviroid based on the observation of the single 250-nt band in the RT-PCR test with CbVds-P1/P2 primers. Two of these CbVd-1 positive plants (cvs Lovebird and Nutmeg) were also positive for CbVd-1 based on the presence of a single 150-nt band in the RT-PCR assay with CbVd1-F1/R1 primers. One plant (cv Jigsaw) was positive for CbVd-1, i.e. showing the 150-nt band in RT-PCR with CbVd1-F1/R1 primers, but did not show the ca. 250-bp band in RT-PCR with primers CbVds-P1/P2. None of the tested plants were positive for CbVd-5, either with the specific, or universal primers. All coleviroid- and CbVd-1-specific PCR products were sequenced directly using the Sanger methodology, and revealed whole genomes for five isolates of CbVd-1 from Oregon, U.S.A. The genomes of the five CbVd-1 isolates displayed 96.9-100% identity among each other and 96.0-100% identity to the CbVd-1 sequences available in GenBank. Because the sequences from cvs Lovebird, Marrakesh, and Nutmeg, were found 100% identical, one sequence was deposited in GenBank (MZ326145). Two other sequences, from cvs Fire Mountain and Smokey Rose, were deposited in the GenBank under accession numbers MZ326144 and MZ326146, respectively. To the best of our knowledge, this is the first report of CbVd-1 in the United States.

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