scholarly journals Relative and Absolute Quantitative Real-Time PCR-Based Quantifications ofhcnCandphlDGene Transcripts in Natural Soil Spiked withPseudomonassp. Strain LBUM300

2010 ◽  
Vol 77 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Nadine J. DeCoste ◽  
Vijay J. Gadkar ◽  
Martin Filion
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Transcriptional Analysis ◽  
Relative Quantification ◽  
Field Soil ◽  
Quantitative Real Time Pcr ◽  
Qrt Pcr ◽  
The Absolute ◽  
Bacterial Concentrations

ABSTRACTTranscriptional analysis of microbial gene expression using relative quantitative real-time PCR (qRT-PCR) has been hampered by various technical problems. One such problem is the unavailability of an exogenous standard robust enough for use in a complex matrix like soil. To circumvent this technical issue, we made use of a recently developed artificial RNA (myIC) as an exogenous “spike-in” control. Nonsterile field soil was inoculated with various concentrations of the test bacteriumPseudomonassp. strain LBUM300, ranging from 4.3- to 8.3-log bacterial cells per gram of soil. Total soil RNA was extracted at days 0, 7, and 14 postinoculation, and using two-step TaqMan assays,phlD(encoding the production of 2,4-diacetylphloroglucinol) andhcnC(encoding the production of hydrogen cyanide) gene expression was monitored. For relative quantification, a defined quantity ofin vitro-synthesized myIC RNA was spiked during the RNA extraction procedure. Absolute qRT-PCR was also performed in parallel. Both the absolute and relative quantifications showed similar transcriptional trends. Overall, the transcriptional activity ofphlDandhcnCchanged over time and with respect to the bacterial concentrations used. Transcripts of thephlDandhcnCgenes were detected for all five bacterial concentrations, but thephlDtranscript copy numbers detected were lower than those detected forhcnC, regardless of the initial bacterial concentration or sampling date. For quantifying a low number of transcripts, the relative method was more reliable than the absolute method. This study demonstrates for the first time the use of a relative quantification approach to quantifying microbial gene transcripts from field soil using an exogenous spike-in control.

Gene Expression Profiling in Multiple Myeloma Cells Treated with the Novel Anti-Myeloma Agents Zoledronate and Fluvastatin.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5078-5078
Author(s):  
Timothy J. Molloy ◽  
Baulch-Brown Cindy ◽  
Yi-Mo Deng ◽  
Andrew Spencer ◽  
David F. Ma
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Real Time ◽  
Real Time Pcr ◽  
Mevalonate Pathway ◽  
Annexin V ◽  
Quantitative Real Time Pcr ◽  
Myeloma Cells ◽  
Qrt Pcr

Abstract We have shown in vitro that multiple myeloma (MM) cells can be destroyed by treating them with the mevalonate pathway inhibitors zoledronate and fluvastatin. While the efficacy of these compounds singly and combination have been demonstrated, their exact modes of action remain largely unknown. The present study aimed to use microarray and quantitative real-time PCR (QRT-PCR) techniques to analyse gene expression in treated myeloma cells to identify novel genes and pathways involved in the anti-myeloma action of these compounds. The human MM cell line NCI-H929 was treated with zoledronate and fluvastatin singly and in combination, and RNA was extracted and used to interrogate oligonucleotide microarrays consisting of 19,000 features representing known and unknown genes. Quantitative real-time PCR was subsequently used to confirm the expression of several genes of interest. Flow cytometry with Annexin V FITC staining was used to detect apoptosis. It was observed that genes related to apoptosis (caspases and p53-related genes), cell cycle control (cyclins), GTPase signalling (Rabs), and growth and proliferation (growth factors) were particularly affected by zoledronate and fluvastatin, and some of these genetic effects were synergistic when a combination of zoledronate and fluvastatin was used. QRT-PCR confirmed the effects on the caspase- and p53-related apoptotic pathways, and these effects were correlated with increased apoptosis in the myeloma cells. The mevalonate pathway inhibitors fluvastatin and zoledronate are highly efficient at killing MM cells, and their effects appear to be synergistic. Our microarray and QT-PCR analyses demonstrated that the expression of specific groups of genes important to the survival and proliferation of myeloma cells are affected by these compounds. p53 and caspase-dependent pathways appear to be the key apoptotic cascades stimulated. Insights into the mechanisms of these novel therapeutics are important as they might help to define their roles in the treatment of multiple myeloma.

scholarly journals Selection of reference genes for quantitative real-time PCR analysis in cucumber (Cucumis sativus L.), pumpkin (Cucurbita moschata Duch.) and cucumber–pumpkin grafted plants

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6536 ◽  
Author(s):  
Li Miao ◽  
Xing Qin ◽  
Lihong Gao ◽  
Qing Li ◽  
Shuzhen Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Stable Expression ◽  
Quantitative Real Time Pcr ◽  
Graft Union ◽  
Expression Levels ◽  
Qrt Pcr

Background Quantitative real-time PCR (qRT-PCR) is a commonly used high-throughput technique to measure mRNA transcript levels. The accuracy of this evaluation of gene expression depends on the use of optimal reference genes. Cucumber–pumpkin grafted plants, made by grafting a cucumber scion onto pumpkin rootstock, are superior to either parent plant, as grafting conveys many advantages. However, although many reliable reference genes have been identified in both cucumber and pumpkin, none have been obtained for cucumber–pumpkin grafted plants. Methods In this work, 12 candidate reference genes, including eight traditional genes and four novel genes identified from our transcriptome data, were selected to assess their expression stability. Their expression levels in 25 samples, including three cucumber and three pumpkin samples from different organs, and 19 cucumber–pumpkin grafted samples from different organs, conditions, and varieties, were analyzed by qRT-PCR, and the stability of their expression was assessed by the comparative ΔCt method, geNorm, NormFinder, BestKeeper, and RefFinder. Results The results showed that the most suitable reference gene varied dependent on the organs, conditions, and varieties. CACS and 40SRPS8 were the most stable reference genes for all samples in our research. TIP41 and CACS showed the most stable expression in different cucumber organs, TIP41 and PP2A were the optimal reference genes in pumpkin organs, and CACS and 40SRPS8 were the most stable genes in all grafted cucumber samples. However, the optimal reference gene varied under different conditions. CACS and 40SRPS8 were the best combination of genes in different organs of cucumber–pumpkin grafted plants, TUA and RPL36Aa were the most stable in the graft union under cold stress, LEA26 and ARF showed the most stable expression in the graft union during the healing process, and TIP41 and PP2A were the most stable across different varieties of cucumber–pumpkin grafted plants. The use of LEA26, ARF and LEA26+ARF as reference genes were further verified by analyzing the expression levels of csaCYCD3;1, csaRUL, cmoRUL, and cmoPIN in the graft union at different time points after grafting. Discussion This work is the first report of appropriate reference genes in grafted cucumber plants and provides useful information for the study of gene expression and molecular mechanisms in cucumber–pumpkin grafted plants.

scholarly journals Evaluation and Validation of Reference Genes for Quantitative Real-Time PCR in Helopeltis theivora Waterhouse (Hemiptera: Miridae)

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Zheng Wang ◽  
Qianqian Meng ◽  
Xi Zhu ◽  
Shiwei Sun ◽  
Shengfeng Gao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Developmental Stages ◽  
Transcriptional Level ◽  
Quantitative Real Time Pcr ◽  
Systematic Analysis ◽  
Helopeltis Theivora ◽  
Qrt Pcr

Abstract Helopeltis theivora Waterhouse is a predominant sucking pest in many tropic economic crops, such as tea, cocoa and coffee. Quantitative real-time PCR (qRT-PCR) is one of the most powerful tools to analyze the gene expression level and investigate the mechanism of insect physiology at transcriptional level. Gene expression studies utilizing qRT-PCR have been applied to numerous insects so far. However, no universal reference genes could be used for H. theivora. To obtain accurate and reliable normalized data in H. theivora, twelve candidate reference genes were examined under different tissues, developmental stages and sexes by using geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder algorithms, respectively. The results revealed that the ideal reference genes differed across the treatments, and the consensus rankings generated from stability values provided by these programs suggested a combination of two genes for normalization. To be specific, RPS3A and Actin were the best suitable reference genes for tissues, RPL13A and GAPDH were suitable for developmental stages, EF1α and RPL13A were suitable for sexes, and RPL13A and RPS3A were suitable for all samples. This study represents the first systematic analysis of reference genes for qRT-PCR experiments in H. theivora, and the results can provide a credible normalization for qRT-PCR data, facilitating transcript profiling studies of functional genes in this insect.

scholarly journals Immune-Related Gene Expression Profile in Laboratory Common Marmosets Assessed by an Accurate Quantitative Real-Time PCR Using Selected Reference Genes

PLoS ONE ◽  
2013 ◽  
Vol 8 (2) ◽  
pp. e56296 ◽  
Author(s):  
Yoshiki Fujii ◽  
Kazutaka Kitaura ◽  
Takaji Matsutani ◽  
Kenji Shirai ◽  
Satsuki Suzuki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Related Gene ◽  
Quantitative Real Time Pcr ◽  
Common Marmosets ◽  
Immune Related Gene

scholarly journals Selection and validation of reference genes for gene expression studies in Klebsiella pneumoniae using Reverse Transcription Quantitative real-time PCR

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Ana Érika Inácio Gomes ◽  
Leonardo Prado Stuchi ◽  
Nathália Maria Gonçalves Siqueira ◽  
João Batista Henrique ◽  
Renato Vicentini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Klebsiella Pneumoniae ◽  
Real Time ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Quantitative Real Time Pcr ◽  
Expression Studies ◽  
Gene Expression Studies
Sign in / Sign up

Export Citation Format

Share Document