Telomere length varies by DNA extraction method: implications for epidemiologic research

Abstract

Background: Both shorter and longer telomeres in peripheral blood leukocyte (PBL) DNA have been associated with cancer risk. However, associations remain inconsistent across studies of the same cancer type. This study compares DNA preparation methods to determine telomere length from patients with colorectal cancer.

Methods: We examined PBL relative telomere length (RTL) measured by quantitative PCR (qPCR) in 1,033 patients with colorectal cancer and 2,952 healthy controls. DNA was extracted with phenol/chloroform, PureGene, or QIAamp.

Results: We observed differences in RTL depending on DNA extraction method (P < 0.001). Phenol/chloroform-extracted DNA had a mean RTL (T/S ratio) of 0.78 (range 0.01-6.54) compared with PureGene-extracted DNA (mean RTL of 0.75; range 0.00-12.33). DNA extracted by QIAamp yielded a mean RTL of 0.38 (range 0.02-3.69). We subsequently compared RTL measured by qPCR from an independent set of 20 colorectal cancer cases and 24 normal controls in PBL DNA extracted by each of the three extraction methods. The range of RTL measured by qPCR from QIAamp-extracted DNA (0.17-0.58) was less than from either PureGene or phenol/chloroform (ranges, 0.04-2.67 and 0.32-2.81, respectively).

Conclusions: RTL measured by qPCR from QIAamp-extracted DNA was less than from either PureGene or phenol/chloroform (P < 0.001).

Impact: Differences in DNA extraction method may contribute to the discrepancies between studies seeking to find an association between the risk of cancer or other diseases and RTL.

Figure 1

Distribution of telomere length by extraction method in the test and validation samples. A) In a test sample, telomere length was measured in 1,033 cases and 2,952 controls whose DNA had been extracted using either phenol/chloroform, PureGene or QIAamp. B) In a validation sample, DNA was extracted from frozen buffy coats of 20 cases and 24 controls using all three extraction methods (phenol/chloroform, PureGene and QIAamp). RTL was measured for each of the DNAs extracted by these different methods. The y-axis shows the telomere length in terms of the log scale of the Telomere/Single copy gene ratio (T/S).

Figure 2

Association between RTL and Cancer Risk: Forest plot clustered by DNA extraction methods. Colored bars indicated the odds ratios (OR) for studies using qPCR to measure relative telomere length (RTL). The blue bars represent RTL results from DNA extracted using column methods, green bars from DNA extracted via salting out methods, and red bars represent RTL from DNA extracted by Phenol/Chloroform. The black bar represents OR for studies using Q-FISH to measure telomere length directly and does not require DNA extraction. Br: breast cancer; CRC: colorectal cancer; Bl: urinary bladder cancer; Mel: melanoma; SCC: squamous cell skin carcinoma; BCC: basal cell carcinoma; Lng: lung cancer; Gas: gastric cancer; Eso: esophageal cancer; NHL: non-Hodgkin's lymphoma; Ren: renal cancer; Pan: pancreatic cancer; Osteo: osteosarcoma; Pros: prostate cancer; Ov: ovarian cancer; Endo: endometrial cancer. *compared quintiles; **compared tertiles; ***compared dichotomized telomere length