Material and Method
RNA isolation, cDNA synthesis and RT-PCR.
RNA was isolated from cultured cells using Trizol® according to the manufacture's protocol. Concentration of RNA was determined spectrophometrically by absorbance at 260 nm with GeneQuant pro (Biochrom Ltd.). First strand complementary DNA was synthesized from 2 μg of total RNA using a commercial revertAid H minus first strand cDNA synthesis kit (Fermentas), according to the manual's instructions. Gene expressionwas determined by quantitative real-time PCR (qRT-PCR), performed in an iCycler IQ detection system (Bio-Rad) using manufacturer's IQ SYBR Green as a double strand DNA-specific fluorescent binding dye. RT-PCR was performed on a final volume of 20 μl, containing 3 μl cDNA (diluted 1:20), 20 pmol of each primer and 10 μl of IQ SYBR® Green Supermix (Bio-Rad, Herlev, Denmark) (100 mM KCl, 40 mM Tris–HCl at pH 8.4, 0.4 mM of each dNTP (dATP, dCTP, and dTTP), iTaq DNA polymerase, 50 U/ml, 6mMMgCl2, SYBR Green I, 20nMfluorescein, and stabilizers). Amplification; 1 cycle for initial denaturation at 95 °C for 15 min, 35 cycles of denaturation, annealing and extension at 30 s at 95 °C, 30 s at 60 °C, 1 min at 72 °C, respectively, and the last cycle for final extension at 72 °C for 7 min. Quantification of target gene and reference gene (housekeeping gene) was performed in duplicate reactions. Primers sequence is available in supplementary Table S2.
Alkaline phosphatase activity assay
Alkaline Phosphatase (ALP) activity assay was carried on whole cell extracts using a colorimetric method(34). Briefly, ALP activity assay was performed using p-nitrophenyl phosphate as a substrate (ABX Pentra ALP CP kit, HORIBA ABX Diagnostic, Montpellier, France). ALP activity was normalized to total cellular protein assessed by the Bradford assay (Bio-Rad) and expressed as units/mg protein. One unit of alkaline phosphatase activity is defined as the enzyme activity that will liberate 1 μM of p-nitrophenol per 30 min at 37°C.
Telomerase Repeat Amplification Protocol (TRAP)
TRAP is an extensively used method for telomerase activitymeasurements. TRAP was performed as described previously1. There are other methods which are all modifications of the original method2however, all employ the same basic concept. In brief, protein extracts of MEF cells were prepared and protein quantity was measured by Bradford method. TRAP by PCR is a two step process; first, the use of a substrate forward primer (TS-5-´AATCCGTCGAGCAGAGTT-3´) in a reaction mixture, having SYBR® Green Supermix (Bio-Rad, Herlev, Denmark) (100 mM KCl, 40 mM Tris–HCl at pH 8.4, 0.4 mM of each dNTP (dATP, dCTP, and dTTP), iTaq DNA polymerase 50 U/ml, 6mMMgCl2, SYBR Green I, 20nMfluorescein, and stabilizers) and incubated for 30 – 40 minutes at 25oC. This time and temperature favours endogenous telomerase activity to add denovo repeats (TTAGGG) to the substrate. Secondly, denovo repeats by telomerase were amplified by reverse ACX (reverse primer, especially designed primer to avoid primer dimer artefacts which are rarely observed but can easily be distinguishable from a 6 bp TRAP ladder) primer (5´-GCGCGGCTTACCCTTACCCTTACCCTAACC-3´) utilizing real time PCR; 24 cycles of 95oC for 30sec, 52oC for 30sec and 72oC for 30sec. EGTA in the reaction mixture was added to improve the amplification of the threshold cycle (Ct). Negative control samples were prepared by utilizing 5µg of RNase at 37oC for 20 min or at 85oC for 10 minutes before the telomerase assay. Positive control samples containing telomerase activity were selected (Human bone marrow stromal stem cells immortalized by telomerase, hTERT). Standard curve was made by 1:5 dilution series of positive control samples. The relative telomerase activity was calculated using following equation; RTA= 10((Ct sample – Yint)/slope).
Fig. 1 S. Surface marker profiles of BMSC and MEF. Cell surface marker profiles were obtained by utilizing Immuno-cytochemical staining.BMSC and MEF were immune-stained for CD surface markers as described in method section. Bar: (E) 20µm
Table1S. List of primers used in real time PCR (mouse)Construct / Forward sequence / Reverse sequence / Product size
Ostoeblast specific
mRunx2 / 5′- ATT TAG GGC GCA TTC CTC ATC -3′ / 5′- TGT AAT CTG ACT CTG TCC TTG TGG AT -3′ / 116
mOsterix / 5′- TAT GCT CCG ACC TCC TCA AC -3′ / 5′- AAT AGG ATT GGG AAG CAG AAA G -3′ / 120
mAlpl / 5′- GCC CTC TCC AAG ACA TAT A -3′ / 5′- CGG TGG CGA GGT GGT CCC AT -3′ / 373
mCol1a1 / 5′- GGT GAA CAG GGT GTT CCT GG -3′ / 5′- TTC GCA CCA GGT TGG CCA TC -3′ / 503
mIbsp / 5′- CAG AAG TGG ATG AAA ACG AG-3′ / 5′- CGG TGG CGA GGT GGT CCC AT -3′ / 257
mOpn / 5′- GAA ACT CTT CCA AGC AAT TC -3′ / 5′- GGA CTA GCT TGT CCT TGT GG -3′ / 589
mSparc / 5-´CCCTTGTTTAAAATGTTTGG-3´ / 5´-TGGGAGTCTGTCATGTGTGC-3´ / 528
mOC / 5´-TGC GCT CTG TCT CTC TGA CC-3´ / 5´-CTG TGA CAT CCA TAC TTG CAG G-3´ / 357
Adipocyte specific
mPpar γ / 5′- GGG TCA GCT CTT GTG AAT GG -3′ / 5′- CTG ATG CAC TGC CTA TGA GC -3′ / 212
mC/EBPα / 5′- AAG CCA AGA AGT CGG TGG A -3′ / 5′- CAG TCC ACG GCT CAG CTG TTC -3′ / 188
mAdipoq / 5′- GAC GTT ACT ACA ACT GAA GAG C -3′ / 5′- CAT TCT TTT CCT GAT ACT GGT C -3′ / 532
maP2 / 5′- CAA AAT GTG TGA TGC CTT TGT G -3′ / 5′- CTC TTC CTT TGG CTC ATG CC -3′ / 416
Chondrocyte specific
mSox9 / 5′- CGA CTA CGC TGA CCA TCA GA -3′ / 5′- AGA CTG GTT GTT CCC AGT GC -3′ / 188
mCol2a1 / 5′- ACA TGT CAG CCT TTG CTG GC -3′ / 5′- CAT GGT CTC TCC AAA CCA GA -3′ / 404
mAcan / 5′- CCC GGT ACC CTA CAG AGA CA-3′ / 5′- ACA GTG ACC CTG GAA CTT GG -3′ / 203
mCol10a1 / 5′- CCT GCA GCA AAG GAA AAC TC -3′ / 5′- TGG CTT AGG AGT GGG AGC TA –3′ / 179
Reference gene
mB-actin / 5´-CAG CTT CTT TGC AGC TCC TT-3´ / 5´-CAC GAT GGA GGG GAA TAC AG-3´ / 157
Reference List
1. Herbert,B.S., A.E.Hochreiter, W.E.Wright, and J.W.Shay. 2006. Nonradioactive detection of telomerase activity using the telomeric repeat amplification protocol. Nat. Protoc. 1:1583
85-1590.
2. Kim,N.W., M.A.Piatyszek, K.R.Prowse, C.B.Harley, M.D.West, P.L.Ho, G.M.Coviello, W.E.Wright, S.L.Weinrich, and J.W.Shay. 1994. Specific association of human telomerase activity with immortal cells and cancer. Science 266:2011-2015.