Determination of Absolute Telomere Length in Cattle and the Effect of DNA Extraction

doi:10.13523/j.cb.2211008

China Biotechnology

2023, Vol. 43

Issue (8): 63-71 DOI: 10.13523/j.cb.2211008

Determination of Absolute Telomere Length in Cattle and the Effect of DNA Extraction

BAO Li-wen^1**(

),CAI Qin^1**,ZHOU Yi-ye^1,^2,³,XU Miao^1,³,SHU Juan^1,³,LI Hua^1,³,LI Hong-yu^1,³,ZENG Yi-tao^1,³,ZENG Fanyi^1,^2,^3,^***(

)

1 Shanghai Institute of Medical Genetics, Shanghai Children’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200040, China
2 Department of Histo-Embryology, Genetics and Developmental Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
3 Key Laboratory of Embryo Molecular Biology, Ministry of Health and Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China

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Abstract

Objective: Telomere is a highly conserved structure at the end of eukaryotic chromosomes responsible for maintaining chromosomal stability, and the length of its DNA sequence, known as telomere length, gradually shortens with age or disease development. Telomere length can provide a reference for assessing the aging and health status of individuals. However, there is no satisfactory method for determining the absolute telomere length from a small amount of cattle samples. Real-time quantitative PCR (qPCR) was used to determine the absolute telomere length of minute DNA samples from cows and to assess the effect of the DNA extraction method on the results of absolute telomere length determination. This study aims to provide a reference for selecting suitable DNA extraction methods and telomere length analysis methods for a study on telomere length in cattle. Methods: The absolute values of telomere length were determined by qPCR. DNA was extracted from the same samples with silica membrane, phenol-chloroform, and magnetic beads, and the telomere lengths were analyzed by terminal restriction fragment (TRF) assay and qPCR, respectively, to compare their effect on cattle absolute telomere length determination. Results: (1) qPCR can be used to determine the absolute telomere length of nano-gram level cow DNA samples with good reproducibility and correlates well with the “gold standard” TRF results. (2) DNA extracted by different methods would result in significant differences in results when used for telomere length analysis. DNA extracted by the magnetic bead method showed the best agreement when telomere length was measured by TRF and qPCR methods. Conclusion: qPCR is a more sensitive, convenient, and rapid method for determining absolute telomere length than the TRF method and is suitable for determining minute DNA samples. The DNA extraction method can affect the results of telomere length determination and should be unified while the assay was performed, and the magnetic bead method was optimal.

Key words： Telomere length qPCR TRF DNA extraction methods

Received: 07 November 2022 Published: 05 September 2023

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	Li-wen BAO
	Qin CAI
	Yi-ye ZHOU
	Miao XU
	Juan SHU
	Hua LI
	Hong-yu LI
	Yi-tao ZENG
	Fanyi ZENG

Cite this article:

BAO Li-wen, CAI Qin, ZHOU Yi-ye, XU Miao, SHU Juan, LI Hua, LI Hong-yu, ZENG Yi-tao, ZENG Fanyi. Determination of Absolute Telomere Length in Cattle and the Effect of DNA Extraction. China Biotechnology, 2023, 43(8): 63-71.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2211008 OR https://manu60.magtech.com.cn/biotech/Y2023/V43/I8/63

Fig.1 Standard curves used to calculate absolute telomere length (a) Standard curve for calculating telomere length of per reaction tube (b) Standard curve for calculating ZAR1 copy number

Fig.2 Comparison of the results of telomere length determination in cow ear skin at different ages by qPCR Each point in the graph represents the telomere length of ear skin sample in each cow. Data are expressed as mean ± standard deviation (mean ± SD).^* P<0.05;^** P<0.01;^*** P<0.001

Fig.3 Reproducibility of telomere length measured by qPCR Each point in the graph represents one sample

Table 1 Comparison of the TRF and qPCR methods for telomere length determination

Table 2 Information of cow ear skin biopsy

Table 3 Comparison of three different DNA extraction methods

Fig.4 Integrity characterization of DNA extracted by different methods

Fig.5 Telomere length determined by TRF and qPCR (a) Results of TRF assay (b) Comparison of the results of telomere length determination by TRF assay (c) Comparison of the results of telomere length determination by qPCR. Three groups of DNA were obtained by three DNA extraction methods; M1: Silica membrane method; M2: Phenol-chloroform method; M3: Magnetic beads method. Data are expressed as mean ± standard deviation (mean ± SD). ^* P<0.05;^** P<0.01;^*** P<0.001

Fig.6 Correlation between TRF results and aTL Each point in the graph represents one sample. M1: Silica membrane method; M2: Phenol-chloroform method; M3: Magnetic beads method


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