Real-Time PCR Analysis of Hematopoietic Transcription Factors

Total RNA was extracted by using an illustra RNAspin Mini Isolation Kit (GE Healthcare). Isolated RNAs (4 μg) were subjected to cDNA synthesis with a RevertAid first strand cDNA synthesis kit (Fermentas). Real-time PCR was performed on an ABI StepOne Plus Real-Time PCR System (Applied Biosystems) using SYBR-Green reagents (SensiFAST SYBR Hi-ROX mix, Bioline). The samples were examined in triplicate for each assay and analyzed by using the comparative cycle threshold C_T (∆∆C_T) method [3 (link)]. The expression of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control for cDNA contents. Gene-specific amplification was conducted with the following primer sets: for EKLF, 5’-CGGCAAGAGCTACACCAAG-3’ (sense) and 5’-CCGTGTGTTTCCGGTAGTG-3’ (antisense); for GATA1, 5’-CAGTCTTTCAGGTGTACCC -3’ (sense) and 5’-GAGTGATGAAGGCAGTGCAG-3’ (antisense); for ALAS2, 5’-GCAGCACTCAACAGCAAG-3’ (sense) and 5’-ACAGGACGGCGACAGAAA-3’ (antisense); for PBGD, 5’-CGCCTCCCTCTAGTCTCTGCTTCT-3’ (sense) and 5’- GTTGCCACCACACTGTCCGTCTG-3’ (antisense); for GAPDH, 5’-TGGTATCGTGGAAGGACTCATGAC-3’ (sense) and 5’-ATGCCAGTGAGCTTCCCGTTCAGC-3’ (antisense).