Cell-Based Microarrays by Peter Ertl & Mario Rothbauer

Author:Peter Ertl & Mario Rothbauer
Language: eng
Format: epub
Publisher: Springer New York, New York, NY

3.6 Telomere Length of HSCs Derived from Induced Pluripotent Stem Cells

DNA is isolated from individual EBs from each well of the 96well plate by DNAeasy Kit. The assay is determining relative telomere lengths using quantitative PCR. Each DNA sample is compared to the reference DNA sample with a single gene copy. The absolute telomere length is assessed by introducing an oligomer standard of defined number of TTAGGG repeats [10]. (see Note 6 )

3.6.1 Telomere Standard Curve

Detail description of telomere length analysis published here [10, 11] was tested and adjusted for our iPSCs protocol. In brief, serial dilutions (10×) of an oligomer 14×(TTAGGG) repeats (84 base pairs, MW 26667,2) are used to prepare a standard curve. The number of telomere repeats is assessed as follows: The weight of oligomer is calculated as MW divided by Avogadro’s number: g. The highest concentration standard (60 pg of oligomer per reaction) contains molecules of telomere standard. The amount of telomere sequence is calculated as: 1.36 × 109 × 84 (oligomer length) = 1.18 × 108 kb of telomere sequence.

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