Quantifying miRNA and mRNA Expression

Total RNA was extracted with TRIzol reagent (Invitrogen). cDNA was synthesized using Superscript III (Invitrogen) or the Taqman miR Reverse Transcription kit (Applied Biosystems, Foster City, CA) for miR-181a, miR-181b, miR-140 and U44. qRT-PCR was performed with Taqman gene expression assays (Applied Biosystems) following the manufacturer's protocols. miR-181a, miR-181b and miR-140 expression were normalized to U44. KRAS, NRAS and MAPK1 expression were normalized to GAPDH. The comparative cycle threshold (C_T) method as previously described was used for relative quantification of gene expression [47 (link)].

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Huang X., Schwind S., Santhanam R., Eisfeld A.K., Chiang C.L., Lankenau M., Yu B., Hoellerbauer P., Jin Y., Tarighat S.S., Khalife J., Walker A., Perrotti D., Bloomfield C.D., Wang H., Lee R.J., Lee L.J, & Marcucci G. (2016). Targeting the RAS/MAPK pathway with miR-181a in acute myeloid leukemia. Oncotarget, 7(37), 59273-59286.

Publication 2016

TRIzol reagent Superscript III Taqman miR Reverse Transcription kit Taqman gene expression assays

Assays Cdna Gapdh Gene expression Kras Mapk1 Nras Reverse transcription Trizol

Corresponding Organization : City of Hope

Other organizations : University of Maryland, Baltimore

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Variable analysis

independent variables

MiR-181a, miR-181b, miR-140 expression

dependent variables

KRAS, NRAS and MAPK1 expression

control variables

U44 (for normalizing miR-181a, miR-181b, miR-140 expression)
GAPDH (for normalizing KRAS, NRAS and MAPK1 expression)

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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