figure2e: P1 transfections with shRNA Renilla and doxorubicin...

In GranderLab/miR34a_asRNA_project: The miR34AasRNAproject package facilitates the transparency and reproducibility of the miR34a asRNA project and associated publication.

Description

We utilized the P1 construct where the overlapping region of miR34a HG and miR34a AS is cloned with luciferase downstream of miR34a HG and renilla downstream of miR34a AS. The p1 sequence was previously published in Raver-Shapira, N., et al., Transcriptional activation of miR-34a contributes to p53-mediated apoptosis. Mol Cell, 2007. 26(5): p. 731-43. All cell lines were cultured at 5 (Life Technologies). All growth mediums were supplemented with 10 heat-inactivated FBS and 50 <ce><bc>g/ml of streptomycin and 50 <ce><bc>g/ml of penicillin. 2.5x10^5 HCT116wt cells were seeded in a 12-well plate. After 24hrs these were co-transfected with the P1 construct (25ng) and shRenilla2.1 (250ng) using lipo2000 (Life Technologies) standard protocol. 24hrs post-transfection, doxorubicin treatment was initiated using 0, 300, or 500ng/ml. 24hrs post-treatment, RNA was extracted using the RNeasy mini kit (Qiagen) and subsequently treated with DNase (Ambion Turbo DNA-free, Life Technologies). 500ng RNA was used for cDNA synthesis using MuMLV (Life Technologies) and a 1:1 mix of oligo(dT) and random nanomers. QPCR was carried out using KAPA 2G SYBRGreen (Kapa Biosystems) using the Applied Biosystems 7900HT machine with the cycling conditions: 95 <c2><b0>C for 3 min, 95 <c2><b0>C for 3 s, 60 <c2><b0>C for 30 s.

Format

A tibble with 72 rows and 6 variables:

'Biological Replicate'

Numeric; Indicates the biological replicate of the sample.

treatment

Factor; indicates the concentration of doxorubicin (ng/ml).

shRNA

Factor; Indicates the transfected shRNA.

gene

Factor; indicates the gene that the Ct values correspond to.

Ct1

Numeric; Ct value corresponding to the first technical replicate.

Ct2

Numeric; Ct value corresponding to the 2nd technical replicate.

...

Examples

getData('Figure 2e')

GranderLab/miR34a_asRNA_project documentation built on May 26, 2019, 7:26 a.m.