Real-time quantitative PCR
2 μl of cDNA was quantified in duplicate for each sample using LightCycler 480 SYBR Green I (Roche) on a LightCycler 480 II as per manufacturer instructions. Cycling conditions were: 15 min at 95 °C, 45 cycles of [15 s at 94 °C, 25 s at 58 °C, 20 s at 72 °C]. Melt curve cycles immediately followed and were: 5 s at 95 °C, 1 min at 65 °C and then gradual temperature rise to 97 °C at a rate of 0.11 °C/s followed by 30s at 40 °C. GDF3 levels were normalized to MAPK3 [86] as a reference gene because MAPK3 has been shown not to change with differentiation in contrast to many other standard housekeeping genes such as actin, which change dramatically during the differentiation process [87]. Melt curve analysis was performed to verify primer specificity and all primers were tested in a dilution series before use. Data is displayed as fold change above proliferative condition mRNA levels using 2^(ΔΔCt) values.
Primer sequences were obtained from the MIT/Harvard PrimerBank.
GDF3 fwd: 5’ ATGCAGCCTTATCAACGGCTT
GDF3 rev: 5’ AGGCGCTTTCTCTAATCCCAG
GDF3 PrimerBankID: 6679979a1
MAPK3 fwd: 5’ TCCGCCATGAGAATGTTATAGGC
MAPK3 rev: 5’ GGTGGTGTTGATAAGCAGATTGG
MAPK3 PrimerBankID: 21489933a1