Guidelines for Successful Quantitative Gene Expression in Real- Time qPCR Assays

Rocha, Antônio José; Miranda, Rafael de Souza; Sousa, Antônio J.S.; Silva, André Luis Coelho da

doi:10.5772/65850

Cited by 5 publications

(4 citation statements)

References 22 publications

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“…Reverse transcription-PCRs were performed in a StepOne Plus real-time PCR system (Applied Biosystems) using 7.5 l of FastStart Universal SYBR green Master mix (Roche Diagnostics), 6.9 l of cDNA template, and 300 nM of each gene-specific primer (Table 2) ratio is calculated from the quantitative real-time PCR (RT-qPCR) E value assessed for the three genes in the study (Fig. S4), which were within an acceptable E value range (54), and the C T in each case. cyp51A sequencing and protein analysis.…”

Section: Methodsmentioning

confidence: 99%

New Insights into the Cyp51 Contribution to Azole Resistance in Aspergillus Section Nigri

Pérez-Cantero

López-Fernández

Guarro

et al. 2019

Antimicrob Agents Chemother

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Invasive aspergillosis (IA) is a severe condition mainly caused by Aspergillus fumigatus, although other species of the genus, such as section Nigri members, can also be involved. Voriconazole (VRC) is the recommended treatment for IA; however, the prevalence of azole-resistant Aspergillus isolates has alarmingly increased in recent years, and the underlying resistance mechanisms in non-fumigatus species remain unclear. We have determined the in vitro susceptibility of 36 strains from section Nigri to VRC, posaconazole (POS), and itraconazole (ITC), and we have explored the role of Cyp51A and Cyp51B, both targets of azoles, in azole resistance. The three drugs were highly active; POS displayed the best in vitro activity, while ITC and VRC showed MICs above the established epidemiological cutoff values in 9 and 16% of the strains, respectively. Furthermore, expression studies of cyp51A and cyp51B in control condition and after VRC exposure were performed in 14 strains with different VRC susceptibility. We found higher transcription of cyp51A, which was upregulated upon VRC exposure, but no correlation between MICs and cyp51 transcription levels was observed. In addition, cyp51A sequence analyses revealed nonsynonymous mutations present in both, wild-type and non-wild-type strains of A. niger and A. tubingensis. Nevertheless, a few mutations were exclusively present in non-wild-type A. tubingensis strains. Altogether, our results suggest that azole resistance in section Nigri is not clearly explained by Cyp51A protein alteration or by cyp51 gene upregulation, which indicates that other mechanisms might be involved.

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Section: Methodsmentioning

confidence: 99%

New Insights into the Cyp51 Contribution to Azole Resistance in Aspergillus Section Nigri

Pérez-Cantero

López-Fernández

Guarro

et al. 2019

Antimicrob Agents Chemother

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“…In addition to improved accuracy, sensitivity, and rapidity, one of the principal advantages of the real-time PCR over basic PCR is that this technique provides a reliable quantification relationship between the number of starting target sequences (before the amplification by PCR) and the amount of amplicon accumulated in a particular PCR cycle [ 5 ]. This is of paramount importance for the precise quantification of the target nucleic acids, which is critical for mRNA quantification in gene expression analysis [ 7 ] and the determination of the viral load of a clinical specimen [ 8 ]. Moreover, there is no need for post-PCR processes, thus minimizing the chance of cross-contamination due to previous amplicons [ 5 ].…”

Section: Basic Principlesmentioning

confidence: 99%

Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis

Artika

Dewi

Nainggolan

et al. 2022

Genes

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Successful detection of the first SARS-CoV-2 cases using the real-time polymerase chain reaction (real-time PCR) method reflects the power and usefulness of this technique. Real-time PCR is a variation of the PCR assay to allow monitoring of the PCR progress in actual time. PCR itself is a molecular process used to enzymatically synthesize copies in multiple amounts of a selected DNA region for various purposes. Real-time PCR is currently one of the most powerful molecular approaches and is widely used in biological sciences and medicine because it is quantitative, accurate, sensitive, and rapid. Current applications of real-time PCR include gene expression analysis, mutation detection, detection and quantification of pathogens, detection of genetically modified organisms, detection of allergens, monitoring of microbial degradation, species identification, and determination of parasite fitness. The technique has been used as a gold standard for COVID-19 diagnosis. Modifications of the standard real-time PCR methods have also been developed for particular applications. This review aims to provide an overview of the current applications of the real-time PCR technique, including its role in detecting emerging viruses such as SARS-CoV-2.

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“…Many studies developing a v-qPCR assay describe a detailed optimization of the dyetreatment protocol, while a crucial component for accurate v-qPCR, the amplicon length, is often overlooked or only briefly assessed. Highly efficient qPCRs employ amplicons shorter than 200 base pairs (bp) because the chance of polymerization errors is lower and a quick, accurate and efficient quantification will be enabled (11)(12)(13). Many v-qPCRs are adapted from very efficient qPCR assays that are highly specific for the target DNA, which results typically in v-qPCR assays using the recommended short amplicon lengths.…”

Section: Introductionmentioning

confidence: 99%

A Viability Quantitative PCR Dilemma: Are Longer Amplicons Better?

Holm

Ghesquière

Boon

et al. 2021

Appl Environ Microbiol

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The development of viability qPCR (v-qPCR) has allowed for a more accurate assessment of the viability of a microbial sample by limiting the amplification of DNA from dead cells. Although valuable, v-qPCR is not infallible. One of the most limiting factors for accurate live/dead distinction is the length of the qPCR amplicon used. However, no consensus or guidelines exist for selecting and designing amplicon lengths for optimal results. In this study, a wide range of incrementally increasing amplicon lengths (68-906 bp) was used on live and killed cells of nine bacterial species treated with viability dye (PMA). Increasing amplicon lengths up to approximately 200 bp resulted in increasing quantification cycle (Cq) differences between live and killed cells, while maintaining a good qPCR efficiency. Longer amplicon lengths, up to approximately 400 bp, further increased Cq difference, but at the cost of qPCR efficiency. Above 400 bp, no valuable increase in Cq differences was observed. Importance Viability qPCR (v-qPCR) has evolved to a valuable, mainstream technique for determining the number of viable micro-organisms in samples by qPCR. Amplicon length is known to be positively correlated with the ability to distinguish between live and dead bacteria but is negatively correlated with qPCR efficiency. This trade-off is often not taken into account and might have an impact on the accuracy of v-qPCR data. Currently there is no consensus on the optimal amplicon length. This paper provides methods to determine the optimal amplicon length and suggests an amplicon length range for optimal v-qPCR, taking into consideration the trade-off between qPCR efficiency and live-dead distinction.

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Guidelines for Successful Quantitative Gene Expression in Real- Time qPCR Assays

Cited by 5 publications

References 22 publications

New Insights into the Cyp51 Contribution to Azole Resistance in Aspergillus Section Nigri

New Insights into the Cyp51 Contribution to Azole Resistance in Aspergillus Section Nigri

Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis

A Viability Quantitative PCR Dilemma: Are Longer Amplicons Better?

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