Comparison of a quantitative Real-Time PCR assay and droplet digital PCR for copy number analysis of the CCL4L genes

Bharuthram, Avani; Paximadis, Maria; Picton, Anabela C.P.; Tiemessen, Caroline T.

doi:10.1016/j.meegid.2014.03.028

Cited by 44 publications

(31 citation statements)

References 45 publications

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“…Quantification of circulating microRNA in human lung cancer by both methods showed similar results . Analysis of human CCL4L gene copy number by ddPCR was shown to be a far superior method to real‐time PCR, which was more inaccurate at higher copy numbers (3‐6) than at lower copy numbers (0‐2) …”

Section: Discussionmentioning

confidence: 79%

Porcine endogenous retroviruses: Quantification of the copy number in cell lines, pig breeds, and organs

Fiebig

Fischer

Bähr

et al. 2018

Xenotransplantation

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The determination of the PERV copy number using ddPCR extended previous data showing differences between the pig breeds and between different organs of a single animal. The determination of PERV copy numbers can be used to select animals less likely to transmit PERVs during xenotransplantation. In addition, this method will be of special value when PERV proviruses are to be inactivated by CRISPR/Cas9.

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Section: Discussionmentioning

confidence: 79%

Porcine endogenous retroviruses: Quantification of the copy number in cell lines, pig breeds, and organs

Fiebig

Fischer

Bähr

et al. 2018

Xenotransplantation

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“…12 ddPCR was also superior to real-time quantitative PCR for accurate assessment of CCL4 gene copy number variation. 8 Our work indicates that ddPCR can be used as a molecular analytical tool of precision in measuring the copy numbers of EML4-ALK fusion at the RNA levels in FFPE samples from patients with lung adenocarcinoma.…”

Section: Q10mentioning

confidence: 82%

“…Droplet Digital PCR (ddPCR), a recently developed technique, involves emulsification and PCR amplification inside thousands of small droplets, each droplet containing one or no molecules of target DNA or RNA. 8 Precise and absolute quantification of the number of target DNA or RNA molecules in the reaction is simply achieved by counting the number of positive and negative droplets. 9 The strategy reduces competitive amplification, allowing detection of 0.001% mutant fractions, which is 1000 times lower than real-time PCR.…”

Section: Q4mentioning

confidence: 99%

Droplet Digital PCR for Absolute Quantification of EML4-ALK Gene Rearrangement in Lung Adenocarcinoma

Wang

Yang

et al. 2015

The Journal of Molecular Diagnostics

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Crizotinib treatment significantly prolongs progression-free survival, increases response rates, and improves the quality of life in patients with ALK-positive non-small-cell lung cancer. Droplet Digital PCR (ddPCR), a recently developed technique with high sensitivity and specificity, was used in this study to evaluate the association between the abundance of ALK rearrangements and crizotinib effectiveness. FFPE tissues were obtained from 103 consecutive patients with lung adenocarcinoma. Fluorescent in situ hybridization (FISH) and ddPCR were performed. The results revealed that 14 (13.6%) of the 103 patients were positive by dual-color, break-apart FISH. Three variants (1, 2, and 3) of the EML4-ALK gene rearrangements were detected. Thirteen of 14 ALK-positive cases identified by FISH were confirmed by ddPCR (four with variant 1, two with variant 2, and seven with variant 3). The case missed by ddPCR was identified as KIF5B-ALK gene rearrangement by PCR-based direct sequencing. Sixteen patients were detected with low copy numbers of EML4-ALK gene rearrangement, which failed to meet the positive cutoff point of FISH. Two of them responded well to crizotinib after unsuccessful chemotherapy. Our study indicates that ddPCR can be used as a molecular analytical tool to accurately measure the EML4-ALK rearrangement copy numbers in FFPE samples of lung adenocarcinoma patients.

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“…While the qPCR method has been well established, there are some remaining limitations, such as low sensitivity and the requirements of a reference sample, an endogenous control and a standard curve for absolute quantification. Recently, digital PCR has become widely used for research and clinical applications because of advantages such as high sensitivity, accuracy and reproducibility, and in contrast with qPCR, absolute quantification of nucleic acids without standard curves [18–20]. Digital PCR can be used to detect mutations, analyze copy number variations, and quantify specific nucleic acid species [21]; it has proven useful for the analysis of cancer genetic variations[13], heterogeneous methylation [22], fetal screening [23], biomarker analysis [24], viral detection, and mitochondrial DNA alterations in Alzheimer disease [25] and others [26].…”

Section: Discussionmentioning

confidence: 99%

Both absolute and relative quantification of urinary mRNA are useful for non-invasive diagnosis of acute kidney allograft rejection

et al. 2017

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Urinary mRNA analysis with three-gene set (18S rRNA, CD3ε, and IP-10) has been suggested as a non-invasive biomarker of acute rejection (AR) in kidney transplant recipients using quantitative real-time PCR (qPCR). Application of droplet digital PCR (ddPCR), which has been suggested to provide higher sensitivity, accuracy, and absolute quantification without standard curves, could be a useful method for the quantifying low concentration of urinary mRNA. We investigated the urinary expression of these three genes in Korean patients with kidney transplantation and also evaluated the usefulness of ddPCR. 90 urine samples were collected at time of allograft biopsy in kidney recipients (n = 67) and from patients with stable renal function more than 10 years (n = 23). Absolute quantification with both PCR system showed significant higher mRNA levels of CD3ε and IP-10 in AR patients compared with stable transplants (STA), but there was no difference in 18S rRNA expression across the patient groups. To evaluate discrimination between AR and STA, ROC curve analyses of CTOT-4 formula yielded area under the curve values of 0.72 (95% CI 0.60–0.83) and 0.77 (95% CI 0.66–0.88) for qPCR and ddPCR, respectively. However, 18S normalization of absolute quantification and relative quantification with 18S showed better discrimination of AR from STA than those of the absolute method. Our data indicate that ddPCR system without standard curve would be useful to determine the absolute quantification of urinary mRNA from kidney transplant recipients. However, comparative method also could be useful and convenient in both qPCR and ddPCR analysis.

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Comparison of a quantitative Real-Time PCR assay and droplet digital PCR for copy number analysis of the CCL4L genes

Cited by 44 publications

References 45 publications

Porcine endogenous retroviruses: Quantification of the copy number in cell lines, pig breeds, and organs

Porcine endogenous retroviruses: Quantification of the copy number in cell lines, pig breeds, and organs

Droplet Digital PCR for Absolute Quantification of EML4-ALK Gene Rearrangement in Lung Adenocarcinoma

Both absolute and relative quantification of urinary mRNA are useful for non-invasive diagnosis of acute kidney allograft rejection

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