Telomere Length Analysis
a) Uses the O’Callaghan et al, 2008 quantitative real-time PCR (qPCR) method based on Cawthon’s (2002) qPCR method to determine absolute telomere length (aTL) values.
b) As little as 200ng DNA is required for the qPCR telomere length assay.
c) An oligomer standard generates telomere lengths (aTL) in nucleotide base pair values rather than Cawthon’s relative quantification (T/S) values.
d) Samples are measured in triplicates on the same plate, and then averaged.
e) PCR amplifications are established using a a robotic pipettor to ensure maximum pipetting accuracy.
f) Real-time PCR performed using an instrument with unique rotary design for sensitive and accurate performance to reduce measurement error.
g) In addition to running a non-template control, three established reference DNA controls are also run in triplicate on each plate as positive controls
h) Plate to plate telomere length variation is normalized using the geometric mean of the reference DNA samples.
i) Results, expressed as an average telomere length (aTL), are an actual nucleotide sequence length (kb lengths) making it easier to apply to other biomarkers such as cortisol, inflammatory cytokines or other stress biomarker levels offered for testing in the BCL
NOTE: Avoid freeze/thawing to any sample as it shortens the telomere length after sample collection. Keep samples in the -80 freezer for long-term shortage. Please complete the roster and ship the samples to the Biomarker Core Lab at the beginning of the week to avoid freeze/thaw cycling during transit.
Biomarker Core lab (BCL) now offers:
- Cell separation and cell isolation from blood
a) Isolation of peripheral blood mononuclear cells (PBMC)-most specifically lymphocytes and monocytes, is possible using a Ficol or Ficol-plaque layer from anticoagulant (EDTA) blood specimens.
b) Contact us about separation of other cell types [click here for a complete list of cells that can be separated].
c) Up to 10 mls blood can be processed.
d) Complete the online roster sheet with the specimen name and other details.
e) Ship blood samples chilled or frozen on a Monday or Tuesday to arrive in the BCL by the Friday. Avoid freeze thaw cycles.
Note: Saliva telomere lengths are longer than peripheral mononuclear blood cell (PBMC) telomere lengths but the telomere length in the two cell types is highly correlated as they shorten at equivalent rates across a range of tissues.
- DNA extraction
a) Performed on any specimen (saliva, buccal swabs/brushes, cells, buffy coats or blood).
b) Ship samples chilled or frozen on a Monday or Tuesday to arrive in the BCL by Friday. Avoid freeze thaw cycles.
c) As little as 1mL of saliva; one buccal sample, 5×106 diploid cells, 200uL buffy coat or blood is needed for DNA extraction
c) Complete the online roster sheet with the specimen name and other details.
d) DNA extraction is performed using a Qiagen QIAamp DNA mini kit.
- DNA quantification using picogreen
a) Used for the specific determination of double-stranded DNA concentration.
i) the relative insensitivity of absorbance assays;
ii) the inability to distinguish between DNA and RNA;
iii) measurement interference caused by contaminants commonly found in nucleic acid preparations;
iv) the relatively large contribution of cell free nucleotides, single stranded nucleic acids and proteins to an absorbance signal.
c) Recommended to ensure the number of telomeres being determined in each sample are the same across all samples.
d) Performed on as little as 50 uL DNA received or extracted in the BCL.
e) Complete the online roster sheet with the specimen name and concentration (if known) to provide a starting point for choosing the right standard curve for quantification.
f) DNA quantification performed using the ThermoFisher Scientific Quant-iT dsDNA Assay kit.