Quantification of miR-1827 and target genes

The total RNA was purified by using a miRNeasy miRNA Isolation Kit (Qiagen) as we previously described [25 (link)]. The miR-1827 expression levels were determined by real-time PCR using Taqman primers and Taqman PCR master mixture (Applied Biosystems). The expression of miR-1827 was normalized with the expression of U6 snRNA. To detect the mRNA expression of MDM2 and p53 target genes, cDNA was prepared with random primers using TaqMan reverse transcription kit (Applied Biosystems) as previously described [33 (link), 53 (link)]. Gene expression levels were determined by real-time PCR using Taqman PCR master mixture and primers. The expression of genes was normalized to Actin gene.

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Zhang C., Liu J., Tan C., Yue X., Zhao Y., Peng J., Wang X., Laddha S.V., Chan C.S., Zheng S., Hu W, & Feng Z. (2016). microRNA-1827 represses MDM2 to positively regulate tumor suppressor p53 and suppress tumorigenesis. Oncotarget, 7(8), 8783-8796.

Publication 2016

miRNeasy miRNA Isolation Kit Taqman PCR master mixture TaqMan reverse transcription kit

Actin Cdna Expression genes Gene Isolation Mdm2 Mirna Mrna P53 genes Primers Real time pcr Reverse transcription U6 snrna

Corresponding Organization : Rutgers, The State University of New Jersey

Other organizations : Zhejiang University, Center for Systems Biology

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Variable analysis

independent variables

MiR-1827 expression levels

dependent variables

MRNA expression of MDM2 and p53 target genes

control variables

U6 snRNA expression (for normalizing miR-1827 expression)
Actin gene expression (for normalizing mRNA expression of MDM2 and p53 target genes)

controls

Positive control: Not specified
Negative control: Not specified

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