Total RNAs were isolated from cultured erythroblast cells on days 6, 8 10, 12, and 14 using TRIzol reagent (Invitrogen) and converted intocomplementary DNAs (cDNAs) using SuperScript III reverse transcriptase with oligo-dT primer (Invitrogen).
The synthesized cDNAs were quantified with specific primers for HBS1L transcripts using SYBR master mix (Applied Biosystems) according to the manufacturer’s recommended conditions. Expression of α-, β-, and γ-globin was measured by SYBR green-based qPCR using primer sequences [19 (link)]. Quantitative PCR was performed on CFX96™ Real-Time system (Bio-Rad). The expression of α-, β-, and γ-globin mRNA in shNTC and shHBS1L transduced cells were calculated by 2-ΔΔCt methods relative to untransduced (UNT) control as described below.
In comparison to untransduced cells, the abundance of the mRNAs for the following erythroid-related transcription factors, namely BCL11A, ZBTB7A, KLF1, GATA1, GATA2, MYB, and ATF4, was measured and displayed as a fold change [20 (link)]. The primer sequences utilized in this study are listed inS2 Table .
The synthesized cDNAs were quantified with specific primers for HBS1L transcripts using SYBR master mix (Applied Biosystems) according to the manufacturer’s recommended conditions. Expression of α-, β-, and γ-globin was measured by SYBR green-based qPCR using primer sequences [19 (link)]. Quantitative PCR was performed on CFX96™ Real-Time system (Bio-Rad). The expression of α-, β-, and γ-globin mRNA in shNTC and shHBS1L transduced cells were calculated by 2-ΔΔCt methods relative to untransduced (UNT) control as described below.
In comparison to untransduced cells, the abundance of the mRNAs for the following erythroid-related transcription factors, namely BCL11A, ZBTB7A, KLF1, GATA1, GATA2, MYB, and ATF4, was measured and displayed as a fold change [20 (link)]. The primer sequences utilized in this study are listed in
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