A method for quantification of absolute amounts of nucleic acids by (RT)-PCR and a new mathematical model for data analysis

Vu, Huong; Troubetzkoy, Serge; Nguyen, Huan Huu; Russell, Michael W.; Městecký, Jiří

doi:10.1093/nar/28.7.e18

Cited by 38 publications

(11 citation statements)

References 16 publications

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“…False discovery rate was calculated to correct the p-value. qRT-PCR relative fold change results are calculated using the 2 -∆∆Ct method (19), where [∆Ct = (Ct RNAs -Ct GAPDH ) and ∆∆Ct = ∆Ct tumor tissues -∆Ct adjacent non-tumor tissues ].…”

Section: Discussionmentioning

confidence: 99%

Integrated analysis of long non-coding RNA competing interactions reveals the potential role in progression of human gastric cancer

Liang

Yao

et al. 2016

International Journal of Oncology

112

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Abstract. Abnormal expression of long non-coding RNAs (lncRNAs) have been shown to play an important role in tumor biology. The Cancer Genome Atlas (TCGA) platform is a large sample sequencing database of lncRNAs, and further analysis of the associations between these data and patients' clinical related information can provide new approaches to find the functions of lncRNA. In the present study, 361 RNA sequencing profiles of gastric cancer (GC) patients were selected from TCGA. Then, we constructed the lncRNA-miRNA-mRNA competitive endogenous RNA (ceRNA) network of GC. There were 25 GC specific lncRNAs (fold change >2, p<0.05) identified, 19 of them were included in ceRNA network. Subsequently, we selected these 19 key lncRNAs and analyzed the correlations with clinical features and overall survival, 14 of them were discriminatively expressed with tumor size, tumor grade, TNM stage and lymphatic metastasis (p<0.05). In addition, eight lncRNAs (RPLP0P2, FOXD2-AS1, H19, TINCR, SLC26A4-AS1, SMIM10L2A, SMIM10L2B and SNORD116-4) were found to be significantly associated with overall survival (log-rank p<0.05). Finally, two key lncRNAs HOTAIR and UCA1 were selected for validation of their expression levels in 82 newly diagnosed GC patients by qRT-PCR. Results showed that the fold changes between TCGA and qRT-PCR were 100% in agreement. In addition, we also found that HOTAIR was significantly correlated with tumor size and lymphatic metastasis (p<0.05), and UCA1 was significantly correlated with tumor size, TNM stage and lymphatic metastasis (p<0.05).The clinical relevance of the two lncRNAs and the bioinformatics analysis results were almost the same. Overall, our study showed the GC specific lncRNAs expression patterns and a ceRNA network in GC. Clinical features related to GC specific lncRNAs also suggested these lncRNAs are worthwhile for further study as novel candidate biomarkers for the clinical diagnosis of GC and potential indicators for prognosis. IntroductionNoncoding RNAs (ncRNAs) are transcripts that have no ability of coding proteins, which widely exit in high eukaryotics. According to their characteristics, ncRNAs can be divided into several subtypes including transfer RNA, small nucleolar RNA (snoRNA), ribosomal RNA (rRNA), microRNA (miRNA) and long non-coding RNA (lncRNA). The amount of the ncRNAs transcripts is >98% of the whole genome transcripts and have been suggested to represent transcriptional noise (1). However, more and more evidence indicates that transcriptional output of genome is far more complex than predicted, and suggests new paradigms of ncRNA regulation (2).Recent studies suggest that the ncRNAs may play important biological roles in transcriptional regulation, cellular development, formation of chromosome and RNA modification (3). Based on the transcript size, ncRNAs are grouped into small ncRNAs (<200 bp) and long ncRNAs (>200 bp, up to 100 kb). lncRNA is the functional end-product, and the level of lncRNA expression correlates directly with the level of the active molecule. Thus, ...

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Section: Discussionmentioning

confidence: 99%

Integrated analysis of long non-coding RNA competing interactions reveals the potential role in progression of human gastric cancer

Liang

Yao

et al. 2016

International Journal of Oncology

112

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“…Despite the wide variability of results characteristic of conventional RT-PCR assays (Reinhold et al 2001) and its unreliability as a clinical diagnostic tool (Calogero et al 2000), considerable attention is still focused on conventional, competitive protocols (Gilliland et al 1990) with steady improvements to internal standards, references for data normalisation as well as the introduction of new mathematical models for data analysis (Halford et al 1999, Vu et al 2000.…”

Section: Introductionmentioning

confidence: 99%

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Bustin

2002

Journal of Molecular Endocrinology

2,232

1,674

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The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.

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“…C for 25 s. Data were analyzed using the Pfaffl method and the CT values were normalized to the expression rate of GAPDH as a housekeeping gene (Vu et al, 2000).…”

Section: Quantitative Real-time Pcrmentioning

confidence: 99%

Formulation, characterization, and geno/cytotoxicity studies of galbanic acid-loaded solid lipid nanoparticles

Eskandani

Barar

Dolatabadi

et al. 2015

Pharmaceutical Biology

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Context: Galbanic acid (GBA) is a sesquiterpene coumarin with different medicinal properties and anticancer effects. Objective: To improve the anticancer activities of GBA, in the current study, we aimed to fabricate GBA-loaded solid lipid nanoparticles (GBA-SLNs) and study their biological activities in vitro. Materials and methods: Hot homogenization was used for preparation of GBA-SLNs. The encapsulation efficiency (EE) and drug loading (DL) and in vitro release were determined. MTT, DAPI, DNA fragmentation, comet, and Anexin V apoptosis assays were used to compare the anti-cell proliferation and genotoxicity properties of GBA and GBA-SLNs against A549 cells and HUVEC to detect apoptosis and DNA damage in the final concentration of 100 mM after 48 h treatment. Results: Scanning electron microscopy (SEM) and particle size analysis showed spherical SLNs (92 nm), monodispersed distribution, and zeta potential of À23.39 mV. High EE (498%) and long-term in vitro release were achieved. The stability of GBA-SLNs in aqueous medium was approved after 3 months in terms of size and polydispersity index. GBA was able to inhibit A549 growth with an IC 50 value of 62 mM at 48 h. Although GBA-SLNs could also inhibit the growth rate of A549 cells, the effect is perceived after 48 h, as approved by the quantitative expression of Bcl-xL and Casp 9 genes, and also genotoxicity assays. Conclusion: Long-term apoptotic effect of GBA-SLNs compared with GBA may be due to the accumulation of GBA-SLNs in the tumor site because of deviant tumor pathology. Our data confirmed that SLNs could be exploited for sustained lipophilic GBA delivery.

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A method for quantification of absolute amounts of nucleic acids by (RT)-PCR and a new mathematical model for data analysis

Cited by 38 publications

References 16 publications

Integrated analysis of long non-coding RNA competing interactions reveals the potential role in progression of human gastric cancer

Integrated analysis of long non-coding RNA competing interactions reveals the potential role in progression of human gastric cancer

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Formulation, characterization, and geno/cytotoxicity studies of galbanic acid-loaded solid lipid nanoparticles

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