The expressions of selected genes from the microarray results were validated by
qRT-PCR. Six DEGs from BV173 and HL-60 with the same experimental design were
selected based on either having the same expression directions and/or two
different expression directions (MDM2, CCNE2, MYL9, TGFβI, CDKN1A, and CDKN2A)
to confirm the fold difference. The extracted RNA (1 µg) was converted to cDNA
using the Roche® cDNA Synthesis Kit (Roche, Basel, Switzerland) according to the
manufacturer’s protocol. Primer pairs for the selected genes were designed by
First Base Company (First Base, Kuala Lumpur, Malaysia) (see additional file 1
for the list of primers). The qRT-PCR reaction was run using Light Cycler 480
DNA SYBR Green I Master by Roche (Roche, Basel, Switzerland). Triplicate PCR
reactions for each sample (BV173 and HL-60 cells) were conducted in 96-well PCR
plates in 20 µl final reaction volume as recommended by the manufacturer’s
protocol. The relative fold change in RNA expression was analyzed using the
2−ΔΔCT method, where the average of ΔCT values for the amplicon of interest was normalized to that of two
endogenous gene TATA box binding protein (TBP) and actin beta (ACTB), compared
with control specimens19 (link).
qRT-PCR. Six DEGs from BV173 and HL-60 with the same experimental design were
selected based on either having the same expression directions and/or two
different expression directions (MDM2, CCNE2, MYL9, TGFβI, CDKN1A, and CDKN2A)
to confirm the fold difference. The extracted RNA (1 µg) was converted to cDNA
using the Roche® cDNA Synthesis Kit (Roche, Basel, Switzerland) according to the
manufacturer’s protocol. Primer pairs for the selected genes were designed by
First Base Company (First Base, Kuala Lumpur, Malaysia) (see additional file 1
for the list of primers). The qRT-PCR reaction was run using Light Cycler 480
DNA SYBR Green I Master by Roche (Roche, Basel, Switzerland). Triplicate PCR
reactions for each sample (BV173 and HL-60 cells) were conducted in 96-well PCR
plates in 20 µl final reaction volume as recommended by the manufacturer’s
protocol. The relative fold change in RNA expression was analyzed using the
2−ΔΔCT method, where the average of ΔCT values for the amplicon of interest was normalized to that of two
endogenous gene TATA box binding protein (TBP) and actin beta (ACTB), compared
with control specimens19 (link).
