The Measurement of Telomere Length in Sprague-Dawley Rats by Quantitative PCR

Abstract

Telomere consist of sequential repetitions of guanine-rich short sequences, such as TTAGGG, and some related proteins complexes in the ends of chromosomes. The different organisms and different cells and tissues of same organisms have different telomere lengths. Telomere contributes to the protection of chromosomal stability by shorten ∼50-150 base during DNA replication. Telomere length was measured by several different methods as well as Southern blot, which is considered the gold standard in the studies to understand the importance of telomere in aging, cancer and normal cell biology. These methods has specifc superior and limitations to himself. In this study, we aimed to create a quantitative PCR protocol, we can use to measurement of telomere length of rats and can also be applied in subsequent studies, and to obtain information about the telomere lengths in the different tissues of rats by this protocol. Genomic DNA was extracted from liver, small intestine, pancreas tissue and lymphocytes of four male Sprague-Dawley rat. The 36B4 gene was chosen as single copy gene for determine the number of genom in the samples. The number of genome copies were found in a rat tissue by qPCR that is used the 36B4 gene-specifc primers. This sample, that is used as rat genom standarts, was used for fnd the number of genome in all samples. The qPCR was performed for telomere (T) and 36B4 (S) in all samples. The T/S ratio was calculated by the ratio of the products derived from two studies to each other, and then the relative T/S ratio was calculated by using a calibrator sample for each sample. In addition, telomere lengths of the samples were also calculated as kilobase by using 10,2 kb long telomere standard. We reached to 100% of effciency in the studies of telomere and 36B4 PCR, it is necessary to the relative quantitation. We also demonstrated by the value of slope in ΔCt graphics versus logaritmic initial concentration is less than 0,1. In addition, a statistically any signifcant difference found between tissues of rat for telomere length (p<0.05). In conclusion, we think that we created a sensitive, easy, and quick Q-PCR protocol for the measurement of telomere length in tissues of rats. © 2011 OMÜ Tüm haklari saklidir.