Simple quantitative PCR analysis for allelic Pten loss in tumor progression.
We describe a simple method to accurately detect and quantify both Pten mutation and allele-specific loss using allele-specific PCR analysis. Our approach used a heterozygous genomic DNA with one wild-type and one mutant Pten allele as a reference at a single concentration to calculate the percent ratio of the wild-type Pten gene for the detection of allele-specific gene loss. With a standard curve, ratios from PCR data were used to quantitate the wild-type Pten allele copy number loss in tumor specimens. We demonstrate the utility of our approach to calculate allele-specific Pten loss during tumor progression and show that our approach generates quantitative data that are comparable to those obtained from digital droplet PCR. As a method to detect both mutation and allele-specific gene loss, our approach is less subject to the variability of sample amount that are often very limited in clinical analysis. Since conventional PCR is easy to be carried out, our method simplifies the workflow in any laboratory and would provide significant advantages for simplicity to quantify allele-specific gene loss.
Department of Physiology, Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, China; Department of Pneumology, The First People's Hospital of Lianyungang City, Lianyungang, 222002, China.
Xingping
X
Wu
Department of Physiology, Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, China; Howard University, Dixon Building, Room 210, 600 W. Street NW, Washington DC, 20059, USA.
Ailin
A
Zhu
Howard University, Dixon Building, Room 210, 600 W. Street NW, Washington DC, 20059, USA.
Xiaowu
X
Pang
Department of Physiology, Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, China. Electronic address: Serene_x@163.com.
Guiqin
G
Xie
Howard University, Dixon Building, Room 210, 600 W. Street NW, Washington DC, 20059, USA.
Xinbin
X
Gu
D000483:Alleles; D000818:Animals; D056915:DNA Copy Number Variations / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics*; D018450:Disease Progression; D020868:Gene Silencing; D051379:Mice; D009154:Mutation / Q000235:genetics; D051059:PTEN Phosphohydrolase / Q000172:deficiency* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D013961:Thyroid Gland / Q000378:metabolism*; D013964:Thyroid Neoplasms / Q000235:genetics* / Q000473:pathology
D004273:DNA, Neoplasm; D051059:PTEN Phosphohydrolase; C487757:Pten protein, mouse
Allele-specific PCR; Allele-specific copy number