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The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay

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77 days ago
The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay https://t.co/rBpgMs9pnn https://t.co/vMpeSAZpA6
The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay https://t.co/8qnwiOHjXn https://t.co/ouS9Ahz88D
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Supplemental Information

Fig1. Overview of animal experiments carried out in this study

DOI: 10.7287/peerj.preprints.27925v1/supp-1

Fig2. Amplification plot of blood sample and human cells

Specific amplification was detected in blood samples. There were no amplification in human cells and distilled water (DW).

DOI: 10.7287/peerj.preprints.27925v1/supp-2

Table1. Primer sequences used in this study

DOI: 10.7287/peerj.preprints.27925v1/supp-3

Table2. Detection of transgene fragments in blood samples by TaqMan qPCR and SYBR Green qPCR

Transgene fragments were detected in all samples in TaqMan qPCR. IV is intravenous injection, IM is intramuscular injection, and IP is intraperitoneal injection of the hEPO coding naked plasmid. **: p < 0.01 vs Pre in each group. *: p < 0.05 vs Pre in each group.

DOI: 10.7287/peerj.preprints.27925v1/supp-4

Fig 5. Confirmation of accuracy and specificity in TaqMan qPCR from stool samples using Sanger sequence methods

Single peak was detected only in IV sample, but concordance between hEPO-reference and amplicon sequences in all samples was confirmed. IV is intravenous injection, IM is intramuscular injection, and IP is intraperitoneal injection of hEPO coding naked plasmid. Red lines indicatethe matching sequences between hEPO-reference and amplicon sequences.

DOI: 10.7287/peerj.preprints.27925v1/supp-5

Table3. Detection of transgene fragments in stool samples by TaqMan qPCR and SYBR Green qPCR

Transgene fragments were detected in all samples in TaqMan qPCR. IV is intravenous injection, IM is intramuscular injection, and IP is intraperitoneal injection of hEPO coding naked plasmid. **: p < 0.01 vs Pre in each group. *: p < 0.05 vs Pre in each group.

DOI: 10.7287/peerj.preprints.27925v1/supp-6

Fig4. Confirmation of the accuracy and specificity of TaqMan qPCR from blood samples using Sanger sequence methods

Single peak was detected from all samples and concordances between hEPO-reference and amplicon sequences in all samples was high. IV is intravenous injection, IM is intramuscular injection, and IP is intraperitoneal injection of hEPO coding naked plasmid. Red lines indicatethe matching sequences between hEPO-reference and amplicon sequences.

DOI: 10.7287/peerj.preprints.27925v1/supp-7

Fig3. Confirmation of the accuracy and specificity in TaqMan qPCR using agarose gel electrophoresis

A single band was detected from blood samples, whereas two bands were detected from stool samples. There was no band in the negative control (human cells). IV is intravenous injection, IM is intramuscular injection, and IP is intraperitoneal injection of hEPO coding naked plasmid. HK: HK-2 cells (10 ng/μL), HuH7: HuH-7 cells (10 ng/μL), HEF: Human embryonic fibroblasts cells (10 ng/μL), 293: Human embryonic kidney 293 cells (10 ng/μL), HMS: Human mesenchymal stem cells (10 ng/μL), DW: Distilled water.

DOI: 10.7287/peerj.preprints.27925v1/supp-8

Additional Information

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

Kai Aoki conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.

Takehito Sugasawa conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.

Kouki Yanazawa conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.

Koichi Watanabe contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper.

Tohru Takemasa contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper.

Yoshinori Takeuchi contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper.

Yuichi Aita contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper.

Naoya Yahagi authored or reviewed drafts of the paper.

Yasuko Yoshida contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper.

Tomoaki Kuji authored or reviewed drafts of the paper.

Nanami Sekine authored or reviewed drafts of the paper.

Kaoru Takeuchi contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper.

Haruna Ueda contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper.

Yasushi Kawakami authored or reviewed drafts of the paper.

Kazuhiro Takekoshi authored or reviewed drafts of the paper.

Animal Ethics

The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):

Animal experiments in this study were approved by the Animal Care Committee, University of Tsukuba (approval numbers: 19-163 and 19-425).

Data Deposition

The following information was supplied regarding data availability:

The raw measurements are provided in the supplementary files 1. The raw data shows all measurements data in PCR which showed that Taqman PCR could detect trans gene fragments ,but SYBR PCR could not detect the trans gene fragments. These datas were used for statistical analysis to compare Taqman PCR and SYBR PCR.

Funding

This work was supported by a grant from the promotional business of doping prevention activities, Japan Sports Agency (JSA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


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