Validation of a Quantitative Polymerase Chain Reaction Method for Human Alu Gene Detection in Microchimeric Pigs Used as Donors for Xenotransplantation

Abellaneda, J.M.; Martı́nez-Alarcón, L.; Quereda, J.J.; Herrero-Medrano, J.M.; Mendonça, L.; Mrowiec, Anna; García-Nicolás, Obdulio; Pallarés, F. J.; Rı́os, Antonio; Muñoz, Antonio; Ramı́rez, Pablo; Ramis, G.

doi:10.1016/j.transproceed.2014.11.016

Cited by 6 publications

(4 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In our qRT-PCR experiments, the Ct values were higher in the presence of rat DNA (Figure 4C). A similar phenomenon has been observed in the presence of a high amount of porcine DNA 32 . It is not currently clear why the presence of xenogeneic DNA reduces the ampli cation e ciency of hDNA.…”

Section: Discussionsupporting

confidence: 80%

Application of droplet digital PCR to detect human Alu DNA elements in biodistribution assays in pre-clinical studies using rodents

Kim

et al. 2022

Preprint

View full text Add to dashboard Cite

Biodistribution and engraftment assays for administered human cells in animal organs must be performed to ensure the safety and efficacy of the human cells in preclinical studies. Quantitative real-time polymerase chain reaction (qRT-PCR) is currently the standard method used to detect Alu DNA elements. The recently developed droplet digital PCR (ddPCR) method has been adopted in many biological fields, in which the small quantities of DNA must be detected. In this study, we evaluated whether ddPCR could replace qRT-PCR in biodistribution research. We applied ddPCR to detect human Alu DNA elements using various concentrations of genomic DNA (gDNA) extracted from human mesenchymal stem cells spiked with 100 ng of rat gDNA. We found that ddPCR could specifically detect human DNA and exhibited linearity within a concentration range of 10 ag–100 pg/20 µl. ddPCR was far more sensitive than qRT-PCR in measuring Alu elements, with a reliable lower limit of detection of 100 ag DNA/20 µl reaction. Additionally, the copy numbers of Alu elements were quantified using ddPCR in an ex vivo study examining DNA samples extracted from mouse spleen and lymph nodes that had been spiked with human natural killer cells; no human DNA was detected using qRT-PCR in this assay. Thus, we established a ddPCR method for detecting human gDNA in bio-distribution assays of human cells in rodents in preclinical studies.

show abstract

Section: Discussionsupporting

confidence: 80%

Application of droplet digital PCR to detect human Alu DNA elements in biodistribution assays in pre-clinical studies using rodents

Kim

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…Prigent et al . 28 used an AluYb8 based qPCR method to quantify human progenitor cells transplanted in rodent liver 28 , 38 while the same set of primers were also used in pigs 29 . We have adopted a similar methodology to quantify human cancer cells in the ache mutant and wild-type zebrafish.…”

Section: Resultsmentioning

confidence: 99%

“…Moreover, ALU based transplant DNA quantification methods have been used to quantify tumor load in mammalian xenotransplantation studies 26 – 29 . These methods provide high detection power and accuracy even when considerably lower proportion of human cells are found in host (<1e-4%) as well as with lower DNA amounts (0.1 pg) 28 .…”

Section: Introductionmentioning

confidence: 99%

Development of a novel zebrafish xenograft model in ache mutants using liver cancer cell lines

Avci

Keşküş

Targen

et al. 2018

Sci Rep

View full text Add to dashboard Cite

Acetylcholinesterase (AChE), an enzyme responsible for degradation of acetylcholine, has been identified as a prognostic marker in liver cancer. Although in vivo Ache tumorigenicity assays in mouse are present, no established liver cancer xenograft model in zebrafish using an ache mutant background exists. Herein, we developed an embryonic zebrafish xenograft model using epithelial (Hep3B) and mesenchymal (SKHep1) liver cancer cell lines in wild-type and achesb55 sibling mutant larvae after characterization of cholinesterase expression and activity in cell lines and zebrafish larvae. The comparison of fluorescent signal reflecting tumor size at 3-days post-injection (dpi) revealed an enhanced tumorigenic potential and a reduced migration capacity in cancer cells injected into homozygous achesb55 mutants when compared with the wild-type. Increased tumor load was confirmed using an ALU based tumor DNA quantification method modified for use in genotyped xenotransplanted zebrafish embryos. Confocal microscopy using the Huh7 cells stably expressing GFP helped identify the distribution of tumor cells in larvae. Our results imply that acetylcholine accumulation in the microenvironment directly or indirectly supports tumor growth in liver cancer. Use of this model system for drug screening studies holds potential in discovering new cholinergic targets for treatment of liver cancers.

show abstract

“…Information on DNA extraction method, PCR detection method, primer/probe sequences, and the units for cell quantification was investigated from the literatures on cell quantification using Alu-qPCR ( Supplementary Table 3 [ 46 , [48] , [49] , [50] , [51] , [52] , [53] , [54] , [55] , [56] , [58] , [59] , [60] , [61] , [62] , [63] , [64] , [65] , [66] , [67] , [68] , [69] , [70] , [71] , [72] , [73] , [74] , [75] , [76] , [77] , [78] , [79] , [80] , [81] , [82] , [83] , [84] , [85] , [86] , [87] , [88] ], and the trend was summarized in Fig. 3 .…”

Section: Case Studies On Cell Quantification By Qpcr Techniquesmentioning

confidence: 99%

Biodistribution studies for cell therapy products: Current status and issues

Kamiyama

Naritomi

Moriya

et al. 2021

Regenerative Therapy

View full text Add to dashboard Cite

Information on the biodistribution (BD) of cell therapy products (CTPs) is essential for prediction and assessment of their efficacy and toxicity profiles in non-clinical and clinical studies. To conduct BD studies, it is necessary to understand regulatory requirements, implementation status, and analytical methods. This review aimed at surveying international and Japanese trends concerning the BD study for CTPs and the following subjects were investigated, which were considered particularly important: 1) comparison of guidelines to understand the regulatory status of BD studies in a global setting; 2) case studies of the BD study using databases to understand its current status in cell therapy; 3) case studies on quantitative polymerase chain reaction (qPCR) used primarily in non-clinical BD studies for CTPs; and 4) survey of imaging methods used for non-clinical and clinical BD studies. The results in this review will be a useful resource for implementing BD studies.

show abstract

Validation of a Quantitative Polymerase Chain Reaction Method for Human Alu Gene Detection in Microchimeric Pigs Used as Donors for Xenotransplantation

Cited by 6 publications

References 18 publications

Application of droplet digital PCR to detect human Alu DNA elements in biodistribution assays in pre-clinical studies using rodents

Application of droplet digital PCR to detect human Alu DNA elements in biodistribution assays in pre-clinical studies using rodents

Development of a novel zebrafish xenograft model in ache mutants using liver cancer cell lines

Biodistribution studies for cell therapy products: Current status and issues

Contact Info

Product

Resources

About