mirVana™ miRNA Isolation Kit, with phenol

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- For RNA to dissolve better and to yeild high levels, preheat elution buffer at 95C. - Addition of β-mercapto ethanol in lysis buffer can be skipped because the phenol will do the job of nuclease inactivation. - To capture more amount of mRNA, modify the amount of lysis buffer:ethanol (from 1:1 to 1:1.5) during the binding step.

Protocol tips
- For RNA to dissolve better and to yeild high levels, preheat elution buffer at 95C. - Addition of β-mercapto ethanol in lysis buffer can be skipped because the phenol will do the job of nuclease inactivation. - To capture more amount of mRNA, modify the amount of lysis buffer:ethanol (from 1:1 to 1:1.5) during the binding step.

Publication protocol

Total RNA were extracted from A431 cells using the mirVana miRNA isolation kit (Ambion, Inc., Austin, TX, USA), according to the manufacturer’s instruction. RNA’s concentration was determined by Nanodrop (ND1000) spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE) and its purity was determined by bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). We performed comprehensive expression profiling of mature miRNA using the Affymetrix GeneChip 2.0 miRNA Array (Affymetrix, Santa Clara, CA). Labeling of 1μg of total RNA samples was performed using the FlashTag Biotin RNA labeling kit (Genisphere Inc., Hatfield, PA) according to manufacturer’s instruction. Using an Affymetrix GeneChip Fluidics Station 450, the arrays were washed and stained and then scanned with an Affymetrix GeneChip scanner (3000 7G). The CEL files containing the raw Affymetrix 2.0 microRNA array intensity data were processed using the Bioconductor tools that were used for image analysis, data import, background adjustment, normalization (based on RMA algorithm), summarization, and quality assessment [29,30]. Cancer cell line (no EGF application) and two time points after EGF application (3h and 12 h) were assumed as treatments. For each time points, RNAs were extracted from 3 samples as replications. Differential gene expression analyses were performed using linear regression models in the limma package [31]. For transcript analysis (microarray data), Log2 ratio values of each time point verses control (cancer cell line) were assumed as fold change. Those having a Bayesian t-test with adjusted p-value with false discovery rate <0.1 and fold change ≥ +1.5 or fold change ≤ -1.5 were accepted as significant differentially expressed genes.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for mirVana™ miRNA Isolation Kit, with phenol below.

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