Multiplex time-reducing quantitative polymerase chain reaction assay for determination of telomere length in blood and tissue DNA.
In this paper we describe a multiplex time-reducing quantitative polymerase chain reaction (qPCR) method for determination of telomere length. This multiplex qPCR assay enables two pairs of primers to simultaneously amplify telomere and single copy gene (albumin) templates, thus reducing analysis time and labor compared with the previously established singleplex assay. The chemical composition of the master mix and primers for the telomere and albumin were systematically optimized. The thermal cycling program was designed to ensure complete separation of the melting processes of the telomere and albumin. Semi-log standard curves of DNA concentration versus cycle threshold (C                         (t)) were established, with a linear relationship over an 81-fold DNA concentration range. The well-performed intra-assay (RSD range 2.4-4.7%) and inter-assay (RSD range: 3.1-5.0%) reproducibility were demonstrated to ensure measurement stability. Using wild-type, Lewis lung carcinoma and H22 liver carcinoma C57BL/6 mouse models, significantly different telomere lengths among different DNA samples were not observed in wild-type mice. However, the relative telomere lengths of the tumor DNA in the two strains of tumor-bearing mice were significantly shorter than the lengths in the surrounding non-tumor DNA of tumor-bearing mice and the tissue DNA of wild-type mice. These results suggest that the shortening of telomere lengths may be regarded as an important indicator for cancer control and prevention. Quantification of telomere lengths was further confirmed by the traditional Southern blotting method. This method could be successfully used to reduce the time needed for rapid, precise measurement of telomere lengths in biological samples.
Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
Jingjing
J
Jiao
Jing X
JX
Kang
Rui
R
Tan
Jingdong
J
Wang
Yu
Y
Zhang
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000097:blood / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D016615:Telomere
D017931:DNA Primers; D004247:DNA