SUPPLEMENTAL MATERIALS
Materials and Methods
Choice of plasmids, miRNA mimics and transfection reagent concentrations and time points
for luciferase assays and western blot experiments
The present parameters were chosen on the basis of a preliminary and thorough experimentation
performed with various transfection reagents and nucleic acid amounts in these cellular models. For
all the experiments with Polyfect, Attractene, and Hiperfect, we tested different quantities of
transfection reagents and nucleic acid (plasmid, plasmid + miRNA mimics, miRNA mimics) in
order to get the best transfection efficiency with the least toxicity. In details:
-for Polyfect experiments: according to the manufacturer’s instruction the Polyfect volume for 24plate experiments can vary from 2 to 4 µL/well depending on the cell line used for transfection,
whereas the proposed plasmid quantities are 200, 400 and 600 ng. The following quantities were
tested in our setting experiments: for Polyfect 2 µL, 3 µL and 4 µL, for pmirGLO vector (we used
pmirR_G but it’s reasonable that two vectors couldn’t have different transfection efficiency because
of just one different nucleotide) 200, 400 and 600 ng. Each combination was tested in duplicate. We
evaluated the transfection efficacy through a Luciferase Reporter Assay 24 h after transfection,
focusing on Renilla values, since it represents our positive control for transfection. The
combinations 3 µL of polyfect + 400 ng plasmid and 4 µL+600 ng showed very similar efficiency,
so we decide for the first combination.
-for Attractene: according to manufacturer’s instruction, 1.5 µL Attractene/well (in a 24-well plate)
should be added together with 400 ng and 5 nM of miRNA mimics. However, in literature many
papers are reported to use higher concentrations of miRNA mimics in cotransfection experiments
(although they might use different transfection reagent, such as Lipofectamine 2000): 20 nM 1,2, 25
nM3, 50 nM4 up to 100 nM5. Thus, we performed the following setting experiment: in a 24-well
plate, different combinations of Attractene Reagent (1 µL/1.5 µL) and AllStars Hs Cell Death
siRNA/AllStars Negative Control siRNA (10 nM/20 nM/30 nM) were tested in duplicate, always
together with 350 ng of plasmid (pmiR_G was used for this setting experiments). AllStars Hs Cell
Death siRNA (Qiagen) is a siRNA blend which targets essential human genes and causes cell death
48-96 hours after transfection that can be observed by light microscopy. It is used to evaluate the
best transfection efficiency, that is represented by the combination of transfection reagent + siRNA
showing the highest difference in cell vitality between AllStars Hs Cell Death siRNA and AllStars
Negative Control siRNA. The highest cell death was observed for 1.5 µL Attractene and 30 nM
siRNA, so those doses were chosen for following experiments.
-for Hiperfect: for miRNA mimics transfection experiments in the p60 dishes (for wb analyses), we
followed strictly the manufacturer’s instruction, using 20 µL Hiperfect/dish and 5 nM of miRNA
mimic/negative control (checking the transfection efficiency of this combination only, in a previous
setting experiment with AllStar Hs Cell Death siRNA). We were also interested in using the same
concentration we used for the cotransfection experiments, so we tested 30 nM too.
Concerning the chosen time points, 24 hours are proposed in pmirGLO vector manufacturer’s
instruction and reported in literature several times for luciferase reporter assay about
polymorphisms6,7 and plasmid-miRNA cotransfections8,9. 48 hours for the western blot analyses
were chosen again according to literature10,11.
References
1. Richardson K, Nettleton JA, Rotllan N, et al. Gain-of-function lipoprotein lipase variant
rs13702 modulates lipid traits through disruption of a microRNA-410 seed site. Am J Hum
Genet 2013;92:5--14.
2. Chang SH, Lu YC, Li X, et al. Antagonistic function of the RNA-binding protein HuR and
miR-200b in post-transcriptional regulation of vascular endothelial growth factor-A
expression and angiogenesis. J Biol Chem 2013;288:4908--21.
3. Reuland SN, Smith SM, Bemis LT, et al. MicroRNA-26a is strongly downregulated in
melanoma and induces cell death through repression of silencer of death domains (SODD). J
Invest Dermatol 2013;133:1286--93.
4. Liu J, Lu KH, Liu ZL, et al. MicroRNA-100 is a potential molecular marker of non-small
cell lung cancer and functions as a tumor suppressor by targeting polo-like kinase 1. BMC
Cancer 2012;12:519.
5. Xiao W, Bao ZX, Zhang CY, et al. Upregulation of miR-31* is negatively associated with
recurrent/newly formed oral leukoplakia. PLoS One 2012;7:e38648.
6. Guo H, Deng Q, Wu C, et al. Variations in HSPA1B at 6p21.3 are associated with lung
cancer risk and prognosis in Chinese populations. Cancer Res 2011;71:7576--86.
7. Zhang Z, Yu X, Guo Y, et al. Genetic variant in CASP3 affects promoter activity and risk of
esophageal squamous cell carcinoma. Cancer Sci 2012;103:555--60.
8. Zhang L, Wang X, Chen P. MiR-204 down regulates SIRT1 and reverts SIRT1-induced
epithelial-mesenchymal transition, anoikis resistance and invasion in gastric cancer cells.
BMC Cancer 2013 14;13:290.
9. Zhi F, Zhou G, Shao N, et al. miR-106a-5p inhibits the proliferation and migration of
astrocytoma cells and promotes apoptosis by targeting FASTK. PLoS One 2013
27;8:e72390.
10. Wang K, Bodempudi V, Liu Z, et al. Inhibition of mesothelin as a novel strategy for
targeting cancer cells. PLoS One 2012;7:e33214.
11. Liu H, Wang Y, Li X, et al. Expression and regulatory function of miRNA-182 in triplenegative breast cancer cells through its targeting of profilin 1. Tumour Biol 2013;34:1713-22.