Telomere length assays were determined in the cell line for 1 year
using the method of Harley et al.
22 as modified by
Whikehart et al.
23 Essentially, genomic DNA was isolated
by the classic proteinase K-phenol-chloroform-isoamyl alcohol
method
24 from four 60-mm culture dishes (1 ×
10
6 million cells) and treated with the
restriction enzymes
HinfI and
RsaI (2.5 U of each
restriction enzyme per microgram of DNA incubated at
37
o overnight). These enzymes cleave the entire
DNA genome except for the telomere proper and a small DNA segment near
the telomere (subtelomere). The DNA remaining is referred to as a
telomere restriction fragment (TRF). Equivalent amounts of DNA digests
were placed on 0.6% agarose gels and electrophoresed. The TRFs were
hybridized directly on the gels at 37
oC overnight
with
32P-labeled probes
(CCCTAA)
3 using hybridization fluid composed of
5× SSC, 20 mM NaH
2PO
4, 5×
Denhardt’s solution, 250 μg/ml salmon testes DNA, and 0.1% sodium
dodecyl sulfate. The hybridized signals were exposed to x-ray film for
10 hours at −80°C. Telomere lengths were reported as the average
length of TRFs. The average lengths were calculated according to the
formula: average TRF = Σ (OD
I ×
L
I)/Σ
(OD
I)
22 where
OD
I is the density reading from a grid box
representing 0.5 cm of gel per lane, and L
I is
the telomere length, expressed in centimeters, within each box.
One-kilobase DNA ladders were used as references for the boxes, and the
data were obtained on a gel analysis apparatus (Eagle Eye II;
Stratagene, La Jolla, CA). Aging sample assays also included separate
10-day-old culture telomere assays as a reference.