Quantitative real-time PCR: a powerful ally in cancer research

Mocellin, Simone; Róssi, Carlo; Pilati, Pierluigi; Nitti, Donato; Marincola, Francesco M.

doi:10.1016/s1471-4914(03)00047-9

Cited by 176 publications

(99 citation statements)

References 55 publications

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“…In the search for a cytokine pattern that could predict tumor response to IL-2/peptide-based melanoma patient vaccination, 15 we studied cytokine mRNA abundance in fine-needle aspirate material from metastatic lesions by using quantitative real-time PCR. 16,17 We found that pretreatment IL-10 mRNA levels were significantly higher in responding than in nonresponding tumor lesions, suggesting that high IL-10 levels in the tumor microenvironment might be conducive to an effective immune response.…”

Section: Introductionmentioning

confidence: 82%

IL-10 stimulatory effects on human NK cells explored by gene profile analysis

et al. 2004

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The molecular mechanisms underlying the increase of natural killer (NK) cell anticancer activity mediated by interleukin (IL)-10 have not been elucidated. The aim of this study was to identify potential molecular mediators of IL-10 stimulatory effects by exploring the NK cell gene display induced by this cytokine. Gene profile was determined by high-throughput cDNA microarray and quantitative real-time PCR. In vitro, NK cells resting or conditioned with IL-10 were tested for cytotoxicity, migration and proliferation. IL-10 enhanced mRNA levels of cell activation/cytotoxicity-related genes (eg secretogranin, TIA-1, HMG-1, interferon-inducible genes) not upregulated by IL-2. In line with these findings, IL-10 increased NK cell in vitro cytotoxicity against Daudi cells. Unlike IL-2, IL-10 did not show any significant effect on NK cell in vitro proliferation and migration. However, gene profile analysis showed that IL-10 increased the expression of cell migration-related genes (eg L-selectin, vascular endothelium growth factor receptor-1, plasminogen activator, tissue; formyl peptide receptor, lipoxin A4 receptor), which might support a stimulatory effect not evident with the in vitro functional assay. Overall, gene profiling allowed us to formulate new hypotheses regarding the molecular pathways underlying the stimulatory effects of IL-10 on NK cells, supporting further investigation aimed at defining its role in cancer immune rejection.

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Section: Introductionmentioning

confidence: 82%

IL-10 stimulatory effects on human NK cells explored by gene profile analysis

et al. 2004

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“…This is in marked contrast to previously published data [23] in which 82/82 papillary cancers and 21/23 anaplastic cancers were reported to express 14-3-3σ by immunohistochemistry. Studies have shown that real-time RT-PCR is a reliable technique for the quantification of gene expression [32,33]. In general, the use of immuno-histochemistry as a test of gene expression tends to be significantly affected by the sensitivity and specificity of the antibodies used (polyclonal vs. monoclonal) and the type of tissues examined (frozen vs. formalin fixed).…”

Section: Discussionmentioning

confidence: 99%

Regulation of 14-3-3σ expression in human thyroid carcinoma is epigenetically regulated by aberrant cytosine methylation

Lal¹,

Padmanabha²,

Provenzano³

et al. 2008

Cancer Letters

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Increased 14-3-3σ expression has been observed by immunohistochemistry in papillary and anaplastic tumors, but not follicular thyroid cancers. 14-3-3σ mRNA expression and methylation status was examined in tumor cell lines and primary thyroid tissues using real-time RT-PCR, bisulfite sequencing and methylation-specific PCR. Most of the 27 CpG's in the gene's CpG island were methylated in normal thyroid, TPC-1, NPA, FTC-238 and 2-7, which did not express 14-3-3σ. In contrast, they were unmethylated in KAK-1 and anaplastic lines KAT4 and DRO-90. 14-3-3σ expression was not increased in thyroid carcinomas, the majority of which had a methylated CpG island. In addition, 5-aza-dC treatment increased 14-3-3σ expression in the FTC-238 and NPA cell lines, which had low baseline expression. We conclude 14-3-3σ expression in thyroid carcinomas is regulated by CpG island hypermethylation.

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“…Real-time RT PCR has become a method of choice for the investigation of gene expression in biomedical research (32,33). Nevertheless, a number of problems are still associated with its use (34)(35)(36).…”

Section: Normalization Of Real-time Rt Pcr Datamentioning

confidence: 99%

Gene Expression Studies: How to Obtain Accurate and Reliable Data by Quantitative Real-Time RT PCR / IZUČAVANJE EKSPRESIJE GENA: KAKO DOBITI TAČNE I POUZDANE PODATKE KVANTITATIVNIM RT PCR-OM U REALNOM VREMENU

Nestorov¹,

Matić²,

Elaković³

et al. 2013

Journal of Medical Biochemistry

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Summary: Real-time RT PCR has been recognized as an accurate, reliable and sensitive method for quantifying gene transcription. However, several steps preceding PCR represent critical points and source of inaccuracies. These steps include cell processing, RNA extraction, RNA storage, assessment of RNA concentration and cDNA synthesis. To compensate for potential variability introduced by the procedure, normalization of target gene expression has been established. Accurate normalization has become an absolute prerequisite for the correct quantification of gene expression. Several strategies are in use for the normalization of data, including normalization to sample size, to total RNA or to an internal reference. Among these, the use of housekeeping genes as an internal (endogenous) control is the most common approach. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper reference gene for normalization have become increasingly stringent. The aim of this paper is to discuss the concept of normalization in mRNA quantification, as well as to discuss several statistical algorithms developed to help the validation of potential reference genes. By showing that the use of inappropriate endogenous control might lead to incorrect results and misinterpretation of experimental data, we are joining the creators of Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) in an attempt to convince scientists that proper validation of potential reference genes is an absolute Kratak sadr`aj: RT-PCR je prepoznat kao precizna, pouzdana i osetljiva metoda za kvantifikaciju transkripcije gena. Me|utim, ovoj metodi prethodi nekoliko koraka koji predstavljaju kriti~ne ta~ke i izvor potencijalnih gre{aka. Ovi koraci uklju~uju obradu }elijskog materijala, ekstrakciju ĩ uvanje RNK, odre|ivanje koncentracije RNK i sintezu cDNK. Da bi se kompenzovala potencijalna varijabilnost nastala tokom procedure, uvedena je normalizacija ekspresije ciljnih gena. Precizna normalizacija je postala apsolutni preduslov za ta~nu kvantifikaciju ekspresije gena. Postoji nekoliko strategija za normalizaciju eksperimentalnih podataka, uklju~uju}i normalizaciju u odnosu na veli~inu uzorka, ukupnu RNK ili internu kontrolu (referencu). Kao interna (endogena) kontrola naj~e{}e se koriste geni sa stabilnom ekspresijom. Imaju}i u vidu veliku osetljivost, reproducibilnost i ve liki dinami~ki opseg PCR metode, zahtevi za odgova raju}im referentnim genima koji }e se koristiti za normalizaciju podataka postali su veoma restriktivni. Cilj ovog rada je da razjasni koncept normalizacije i prokomentari{e nekoliko statisti~kih algoritama koji su razvijeni kako bi pomogli u validaciji potencijalnih referentnih gena. Pokazuju}i da kori{}enje neodgovaraju}ih referentnih gena (endogenih kontrola) mo`e da dovede do neta~nih rezultata i pogre{ne interpretacije eksperimentalnih podataka, mi se priklju~ujemo tvorcima uputstva MIQE (eng. Minimum Information for Publication of Quantitati...

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Quantitative real-time PCR: a powerful ally in cancer research

Cited by 176 publications

References 55 publications

IL-10 stimulatory effects on human NK cells explored by gene profile analysis

IL-10 stimulatory effects on human NK cells explored by gene profile analysis

Regulation of 14-3-3σ expression in human thyroid carcinoma is epigenetically regulated by aberrant cytosine methylation

Gene Expression Studies: How to Obtain Accurate and Reliable Data by Quantitative Real-Time RT PCR / IZUČAVANJE EKSPRESIJE GENA: KAKO DOBITI TAČNE I POUZDANE PODATKE KVANTITATIVNIM RT PCR-OM U REALNOM VREMENU

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