Southern blotting was performed as previously described using 3 ug of genomic DNA digested with RsaI/HinfI (37 (link)). Digested DNA was separated on a 0.7% agarose gel, transferred to Hybond N membrane (Amersham), and fixed with UV by a Stratalinker (Stratagene). Telomere signal was detected using a DIG labeled telomere sequence probe followed by chemiluminescence detection methodology from Roche according to the manufacturer’s protocol (Roche). Telomere length was calculated by finding the midpoint of the telomere signal and comparing to DIG labelled size standards run on the same gel.
The telomerase assays were performed as previously described using a quantitative PCR methodology (26 (link)). One thousand cells were used per assay and each assay was performed in quadruplicate and average values were calculated along with standard error. Results were calibrated to a standard curve and made relative to the telomerase activity detected in normal skin keratinocytes for comparison.
Quantitative reverse transcriptase PCR (q-RT-PCR) was performed as described (26 (link)). Briefly, total RNA was extracted from cells grown for one passage without feeders using Trizol Reagent (Invitrogen) according to the manufacturer’s instructions and purified with RNeasy Mini Columns (Qiagen). RNA was reverse transcribed using a Retroscript kit (Ambion) with random decamers as recommended by the manufacturer. Quantitative PCR was performed in triplicate using the ABI PRISM 7900 Sequence Detection System with standard cycling in SYBR-Green PCR Master Mix (Applied Biosystems) with the following primers:
TERC-F, 5′ TCTAACCCTAACTGAGAAGGGCGTAG-3′;
TERC-R, 5′ GTTTGCTCTAGAATGAACGGTGGAAG-3′;
TERT-v1-F, 5′-TGTACTTTGTCAAGGTGGATGTGA-3′;
TERT-v1-R, 5′-GCTGGAGGTCTGTCAAGGTAGAG-3′;
GAPDH-F, 5′-AAGGTCATCCATGACAACTTTG-3′;
GAPDH-R, 5′-GTAGAGGCAGGGATGATGTTCT-3′
Expression levels were normalized to GAPDH and made relative to normal untransduced N-HSK-1 cells.
The telomerase assays were performed as previously described using a quantitative PCR methodology (26 (link)). One thousand cells were used per assay and each assay was performed in quadruplicate and average values were calculated along with standard error. Results were calibrated to a standard curve and made relative to the telomerase activity detected in normal skin keratinocytes for comparison.
Quantitative reverse transcriptase PCR (q-RT-PCR) was performed as described (26 (link)). Briefly, total RNA was extracted from cells grown for one passage without feeders using Trizol Reagent (Invitrogen) according to the manufacturer’s instructions and purified with RNeasy Mini Columns (Qiagen). RNA was reverse transcribed using a Retroscript kit (Ambion) with random decamers as recommended by the manufacturer. Quantitative PCR was performed in triplicate using the ABI PRISM 7900 Sequence Detection System with standard cycling in SYBR-Green PCR Master Mix (Applied Biosystems) with the following primers:
TERC-F, 5′ TCTAACCCTAACTGAGAAGGGCGTAG-3′;
TERC-R, 5′ GTTTGCTCTAGAATGAACGGTGGAAG-3′;
TERT-v1-F, 5′-TGTACTTTGTCAAGGTGGATGTGA-3′;
TERT-v1-R, 5′-GCTGGAGGTCTGTCAAGGTAGAG-3′;
GAPDH-F, 5′-AAGGTCATCCATGACAACTTTG-3′;
GAPDH-R, 5′-GTAGAGGCAGGGATGATGTTCT-3′
Expression levels were normalized to GAPDH and made relative to normal untransduced N-HSK-1 cells.
