A quantitative PCR method for measuring absolute telomere length

We describe a simple and reproducible method to measure absolute telomere length (aTL) using quantitative real-time polymerase chain reaction (qPCR). This method is based on the Cawthon method for relative measurement of telomere length (TL) but modified by introducing an oligomer standard to measure aTL. The method describes the oligomer standards, the generation of the standard curve and the calculations required to calculate aTL from the qPCR data. The necessary controls and performance characteristics of the assay are described in detail and compared relative to other methods for measuring TL. Typical results for this assay for a variety of human tissue samples are provided as well as a troubleshooting schedule. This method allows high throughput measurement of aTL using small amounts of DNA making it amenable for molecular epidemiological studies. Compared to the traditional relative TL qPCR assays, the aTL method described in this protocol enables a more direct comparison of results between experiments within and between laboratories.


Introduction
Telomeres are nucleoprotein structures that cap the ends of chromosomes. The integrity of the telomere structure and its DNA hexamer (TTAGGG)n repeat sequence is critical for the protection of the ends of chromosomes from degradation and in maintaining overall genomic stability [1,2]. The number of DNA hexamer (TTAGGG) n repeats is reduced during each cell division in differentiated cells, and as a consequence telomere length (TL) often decreases in most differentiated cells throughout the lifespan of the organism [1]. Shortening of telomeres can result in telomere end fusions and an increased level of chromosome instability (CIN), which is in turn a key initiating event in numerous cancers (including lung, breast, colon, and prostate cancers, as well as certain leukaemia's) [3][4][5][6][7]. It has been shown that telomere shortening can be accelerated by environmental factors such as psychological and physiological stress, cigarette smoking, obesity and high homocysteine [8][9][10][11][12][13][14]. Efficiency of TL maintenance is also affected by gender [15][16][17]. TL has been shown to be associated prospectively with increased risk of myocardial infarction, coronary artery disease, breast cancer free survival, clear cell renal cell carcinoma survival, post-stroke mortality, dementia and cognitive decline, as well as total survival independent of genetic influences [18][19][20][21][22][23][24].
For all of these reasons there has been a burgeoning interest in measuring TL accurately and efficiently to understand both the fundamental biology of telomere maintenance as well as determining the modifiable dietary and life-style factors that contribute substantially to accelerated TL attrition. A wide range of methods have been developed to measure TL such as (i) the gold standard Terminal Restriction Fragment (TRF) analysis by hybridisation of digested DNA with telomere sequence probes, (ii) Flow-FISH cytometry of cells following hybridisation with fluorescent peptide nucleic acid (PNA) probes, (iii) quantitative fluorescence in situ hybridisation (FISH) with fluorescent telomere PNA probes and (iv) qPCR assay. With the exception of the TRF assay all other methods have the disadvantage of generating a relative measure of TL. The advantage of the qPCR method is that, unlike the TRF assay only small amounts of DNA are required and can easily be performed in high-throughput format which is essential for large epidemiological studies [25,26].
The protocol we describe is a modification to Cawthon's qPCR based relative quantification (Telomere/Single Copy Gene ratio) method by introducing an oligomer standard to generate aTL values. The capability to generate aTL values allows a more direct comparison of results between experiments within and between laboratories. The protocol described here is for human buccal cells or isolated lymphocytes but can be easily adapted to other cell types or species.

Experimental design
The measurement of aTL can be performed in any cell population from which high quality undamaged DNA can be collected. However the possibility of detecting meaningful differences between groups is likely to depend on the purity of the cell populations examined because of the possibility of different replicative histories which may affect TL. This is particularly true when doing in vivo studies with white blood cells because the ratios of lymphocytes, monocytes, basophils and granulocytes can vary greatly between individuals depending on their age and health condition(s). Therefore aTL should be measured in populations of single cell types as much as is practically possible. Other sources of variation could be the proportion of dead/dying cells which may have to be taken into consideration because necrotic or apoptotic cells may have a different TL from viable cells.
As indicated above there are an increasing demographic, nutritional and life-style variables and disease conditions associated with altered TL and these need to be considered when planning comparative in vivo studies. Below is a list of the key variables that should be considered in human studies of aTL [8][9][10][11][12][13][18][19][20][21][22][23][24][27][28][29]: Date of Birth (include maternal and paternal age at birth) Gender History of cancer, cardiovascular disease and neurodegenerative disease Inherited mutations that predispose to degenerative diseases listed above as well as accelerated ageing syndromes (e.g., WRN, ATM and BRCA1 mutations) Medication or recent illness (disease history) Smoking status and history Exposure to chemical carcinogens and radiation e.g., X-ray Indicators of psychological stress Body Mass Index Lifestyle index (e.g. physical activity, alcohol consumption, sunshine exposure) Dietary habits measured using a validated food frequency questionnaire Plasma concentration of B vitamins and homocysteine Markers of oxidative stress (CRP, MDA etc) Genotype

Precautions regarding a qPCR based assay
Real-time quantitative PCR (qPCR) uses fluorescent signal detection to be monitored as the PCR reaction proceeds. This allows initial template levels to be precisely and accurately quantified. PCR is an extremely sensitive method of analysis and cross-contamination can lead to erroneous or false results. Therefore precautions and strict quality control ascertainment must be planned into to each and every experiment (further details can be found in [30]).

Materials
Reagents and Reagent Set-Up Sample collection

Oligomers (standards and primers)
CRITICAL: All oligomers should be HPLC purified; long oligomers (>50 mers) have a high failure rate during synthesis; this means there will be multiple failed sequences which must be removed in order to maintain accuracy of oligomer standards (GeneWorks, Adelaide). All oligomers are diluted in appropriate volume of PCR grade water and stored at -20degC until required. Working stocks of oligomers should be made fresh; dilutions are stable at 4degC for up to 2 weeks. Oligomer sequences are shown in Table 1.

Standard Curves and associated calculations Telomere Standard Curve
A standard curve is established by dilution of known quantities of a synthesised 84 mer oligonucleotide containing only TTAGGG repeats. The number of repeats in each standard is calculated using standard techniques as follows: • The oligomer standard is 84 bp in length (TTAGGG repeated 14 times), with a molecular weight (MW) of 26667.2.
• The amount of telomere sequence in TEL STD A is calculated as: 1.36 × 10 9 × 84 (oligomer length) = 1.18 × 10 8 kb of telomere sequence in TEL STD A.
Plasmid DNA (pBR322) is added to each standard to maintain a constant 20 ng of total DNA per reaction tube. The standard curve was used to measure content of telomeric sequence per sample in kb ( Figure 1).

Single copy gene (SCG) standard
A single copy gene (SCG) is used as a control for amplification for every sample performed and to determine genome copies per sample. The choice of SCG is critical for reliability of results; any change in copy number can substantially impact upon aTL measurements. We routinely use 36B4, which encodes the acidic ribosomal phosphoprotein P0; bglobin is also frequently used. NOTE: Remember that although telomeric DNA sequence is consistent in mammals, the SCG will be different, thus a SCG standard curve and amplicon must be generated for each target species.
CRITICAL: SCG amplification is crucial for the accuracy of the results generated in the aTL qPCR assay; changes in amount of template that is present in each reaction can by affected by pipetting or DNA quantification error. Variation in SCG copy number (CNV) may also occur between individuals and between normal and cancerous cells; CNV can be assessed by using multiple SCG amplicons to generate diploid copies per PCR reaction.

Standard curve for human SCG, 36B4
Genome copy number per reaction is calculated as follows: • The synthesised 36B4 oligomer standard is 75 bp in length with a MW of 23268.1.
• Therefore SCG STD A is equivalent to 2.63 × 10 9 diploid genome copies, because there are two copies of 36B4 per diploid genome.
A standard curve was generated by performing serial dilutions of SCG STD A (10 -1 through to 10 -6 dilution).
Plasmid DNA (pBR322) was added to each standard to maintain a constant 20 ng of total DNA per reaction tube. The standard curve was used to measure diploid genome copies per sample ( Figure 1).

PCR
Power SYBR I mastermix (Applied Biosystems, #4367396). Contains AmpliTaq Gold DNA polymerase, dNTPs, SYBR I Green Dye, opitimised buffers and passive reference dye (ROX) -CAUTION SYBR I Green dye may be carcinogenic when ingested or absorbed into skin. Wear appropriate gloves when working with this solution.

Sample collection
The measurement of aTL can be performed in any cell population from which high quality undamaged DNA can be collected. Below are some examples of collection procedures we routinely use in our research. All manipulations must be carried out in a Biological Safety Cabinet Class II.

Lymphocytes
I. Collect fresh blood by venipuncture into vacutainer blood tubes (LiHep or EDTA). II. Dilute whole blood 1:1 with HBSS and gently invert to mix III. Overlay diluted blood onto 1/3 volume of Ficoll-Paque being careful not to disturb the interface IV. Centrifuge tubes at 400 g for 30 min at 20degC V. Remove lymphocyte layer located at the interface of Ficoll-Paque and dilute plasma into a fresh tube using a Pasteur pipette. Ct Figure 1 Standard curve used to calculate absolute telomere length. C T (cycle threshold) is the number of PCR cycles for which enough SYBR green fluorescence was detected above background. A) Graph shows standard curve for calculating length of telomere sequence per reaction tube. X-axis represents amount of telomere sequence in kb per reaction.

Buccal cells
I. Prior to buccal cell collection rinse mouth twice thoroughly with 100 ml of water. II. Gently rotate a small-headed toothbrush (2 cm head length) 10 times firmly against the inside of the cheek wall in a circular motion. III. The head of each brush is then placed into 10 ml of buffer and rotated repeatedly such that the cells are dislodged and released into buffer producing a cloudy suspension of buccal cells in the buffer. IV. Centrifuge for ten minutes at 100 g. V. Remove supernatant leaving approximately 1 ml of cell suspension and replace with another 5 ml of buccal buffer. Vortex briefly. VI. Centrifuge at 100 g for ten minutes. VII. Remove supernatant and resuspend in 5 mls of buccal buffer. VIII. Vortex briefly and then homogenise for 2-3 minutes in a hand held tissue homogenizer to disaggregate cell clumps. IX. Draw cells up into a syringe (with 18G needle) and pass through a 100 μm nylon filter into a fresh tube. X. Centrifuge at 100 g for 10 mins and remove the supernatant. XI. Resuspend in 1 ml of buccal cell buffer.
XII. Perform viable cell count using haemocytometer and trypan blue XIII. Centrifuge at 100 g for 10 min. PAUSE POINT: Cells can be resuspended in 200 μl PBS and taken through to DNA isolation or if storage required proceed to step XIV. XIV. Discard supernatant and resuspend the cell pellet in freezing media (FBS and 10%DMSO) resuspend at 5 × 10 6     Fragment analysis (TRF) [32]. Telomere lengths are determined by a TRF diagnostic kit (Roche Diagnostics, Australia). For example our studies demonstrated a strong correlation between results for TRF and the qPCR method for aTL (r 2 = 0.75, p < 0.0001) (Figure 2). The TRF measurement for the 1301 B-cell derived lymphoblastoid cell line, commonly used in Flow-FISH analysis of telomere length was reported to be 80 kb [33].
Using the absolute Real-Time PCR method, we measure the average telomere length for 1301 at 70 kb. Telomere lengths, in different cell types, showed good agreement between the reported TRF values and the measures we obtained by the absolute qPCR method ( Figure 2). However, there is a consistent discrepancy between the values obtained by the two methods with the TRF value being approximately 7 kb greater than that observed with the aTL [33][34][35]. It is recognised that the TRF method tends to overestimate telomere length because there is a considerable, highly variable, nontelomeric DNA component within TRFs [36]. In contrast, aTL only detects and amplifies intact TTAGGG sequences ( Figure 3). In addition to TTAGGG, terminal restriction fragments in human DNA contain variable amounts of TTAGGG-like repeat sequences, which are detected as telomere sequence in the TRF assay. These include telomere repeat variants proximal to the telomere and the telomere adjacent sequences (reviewed in [36]). Additionally, as TRF is based on hybridisation the shorter the telomere the lower the hybridisation signal, consequently there is a telomere length threshold below which TRF analysis will not detect telomeric DNA. Although TRF analysis biases towards longer TL, this can be partially corrected by dividing the signal intensity by length in base pairs [37], although this is not always done.
Trouble shooting advice 1. Low quality DNA/low yield. There are several possible causes for low yield/quality DNA. The most common reasons include inappropriate storage of sample following collection, non optimal starting amount (too much or too little) of sample collected, and insufficient cell lysis. Suggestion: Reisolate DNA from starting material.
2. Low/no amplification in PCR (positive controls and standards). There are several possible causing for low/no PCR amplification. Suggestion: Check primer dilutions, you may need to set up fresh working dilutions of primers from oligomer stock.
3. Polymerase not working efficiently. Suggestion: Check PCR cycling to ensure appropriate activation of DNA polymerase is in place; select new aliquot of master mix and repeat PCR.
4. Fluorescent dye not working. Suggestion: Check PCR machine setting to ensure appropriate detection method for SYBR Green is selected; select new aliquot of master mix and repeat PCR.
a. low quality DNA (see above) b. too much/too little DNA used (see Variation in Copy Number for expected results and modify accordingly) 6. Amplification in NTC. It is important that you determine the cause of this amplification; results from plates with NTC amplification should not be used. Suggestion: First check dissociation curve plot to determine likely causes of amplification in NTC; gDNA contamination or primer dimer formation. If cause of amplification in NTC is contamination (usually in the PCR set-up) change water and repeat NTC. Primer dimer formation is another cause of signal in the NTC wells. This amplification can be identified via examination of the dissociation curve; primer dimmer dissociation curves will appear very different to telomere dissociation profiles. Comparison of telomere length measurement methods. Graph represents correlation between the TRF and aTL methods. Telomeres were measured in whole-blood, lymphocytes, mid-rectal biopsies and low and high controls (Telo-assay, Roche) (r 2 = 0.75, p < 0.0001).

Figure 3
Schematic representation of aTL measurement by qPCR. One pair of chromosomes is shown, circles represent centromeres. Regions containing telomere repeats (TTAGGG)n are coloured blue. As shown, telomere lengths can vary between chromosomes and even between the two ends of a single chromosome. The arrows represent the primer pairs used to amplify the telomere sequences. The telomere PCR signal is a measure of telomere length, because the number of telomere primers that can bind the telomeric DNA at the beginning of the PCR is directly proportional to the total summed length of all the telomeres in the cell (adapted from [38]).

Anticipated results
A typical data set from lymphocytes and buccal cells of 18 males and 25 females in a young group (aged 18-31 years), and 25 males and 23 females in an older group (65-75 yrs) is shown in Figure 4. For the young group the lymphocytes had a mean aTL of 97.2 kb/diploid genome (range 35-260); the buccal cells had a mean aTL of 211.2 kb/diploid genome (range 45-594). The lymphocytes from the older group had a mean aTL of 86.6 kb/diploid genome (range 35-174); the buccal cells had a mean aTL of 230 kb/diploid genome (range 33-750).