Thirty-five cycles of PCR were carried out as described in Figure 2 and signal was acquired using Bio-Rad iQ5 software version 2.0. For the multiplex qPCR, HPLC-purified forward and reverse telomere primer pair (final concentration of 300 nM each) and single-copy gene, albumin primer pair (final concentration 300 nM each) were included in each reaction ( Table 1). One nontemplate control and two positive controls were also amplified in duplicates in each run. Triplicates of reference DNA and DNA extracted from blood samples were taken in 1× master mix consisting of 0.5× SYBR Green I (Invitrogen, CA, USA) 0.5 U AmpliTaq Gold DNA polymerase (Applied Biosystems, CA, USA), 10 mM Tris-HCl, 50 mM KCl, 3 mM MgCl2 (Invitrogen), 0.2 mM each dNTP (MBI Fermentas, Hanover, MD, USA), 1 mM DTT (Sigma, Germany), 1 M betaine (Sigma, Germany) and nuclease-free water in a final volume of 25 μl and added into reaction wells of 96-well PCR plate (Axygen, CA, USA) compatible with Real-Time PCR detection system (IQ5, software version 2.0, Bio-Rad Laboratories Inc., CA, USA) and amplified. Reference DNA at concentrations of 120, 40, 13.33, 4.44, 1.48 and 0.74 ng/μl were prepared from standard genomic DNA by serial dilution. Leukocyte TL was measured by MMqPCR method as described by Cawthon with minor modifications. In the current study we compared the relative TL measured by multiplex quantitative PCR with absolute TL measured by Southern blot technique. Comparative studies for validation of qPCR with the gold standard Southern blot technique are limited. Proper optimization of qPCR conditions is of importance to reduce variability. Studies have reported wide range of CVs (2–28%) for measurement of TL by qPCR suggesting that reproducibility is a concern with qPCR. An improved version of this technique monochrome multiplex qPCR (MMqPCR) was established by the same author in which both telomeric DNA and single-copy gene are amplified in a same well of a plate and shows less variability compared with monoplex qPCR and has lesser sample requirement. The telomeric DNA measurement is normalized with a single-copy housekeeping gene (S) amplified in same sample in a different plate and T/S ratio is computed which is a measure of relative TL. The method entails detection of telomeric DNA with fluorescent signals (T) using partially mismatched primers in a 96-well format on real time-quantitative PCR (qPCR) platform. This technique was introduced by RM Cawthon in 2002. The quantitative PCR technique which gives relative TL in short time and requires small amount of DNA (20 ng per reaction) is widely used in epidemiological studies. The flow-FISH method is used to assess TL in subsets of cells separated from fresh blood samples and requires expensive equipment which may not be available in many laboratories and cannot be used to assess TL in tissues and stored samples. The single telomere length analysis method is labor intensive and is again not suitable for testing large number of samples. Usage of this technique is limited in studies involving large number of samples due to the method being time consuming, costly, labor intensive and requiring high concentration of DNA. Southern blot technique measures absolute length of telomere restriction fragments (TRF) and is the gold standard method. However due to large interindividual variability and lack of standardization of measurement methods, usage of TL measurement in clinical settings is limited.ĭifferent techniques have been employed to determine telomere length and include Southern blot analysis, quantitative polymerase chain reaction (PCR), flow-fluorescence in situ hybridization (FISH) and single telomere length analysis method. They also play an important role in the progression of cancer. The accelerated telomere attrition is associated with increased risk for several age-related diseases such as cardiovascular disease, diabetes and Alzheimer's dementia. Lifestyle factors can modulate telomere length (TL). The gradual loss of telomeric DNA contributes to the essential cellular senescence and programmed cell death which is associated with normal cellular aging. Telomeres become shorter with every somatic cell division due to incomplete DNA replication, resulting in shortening of telomeric DNA up to 20–200 bp with each cell division. This nucleotide sequence (5′- TTAGGG-3′, in human) repeats approximately 2500-times but shows intraindividual variation and differs with different stages of growth. Telomeres are tandemly repeated hexa-nucleotide sequence at the ends of chromosomes that protect the coding DNA by capping chromosomal ends and together with associated proteins.
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