TRIzol Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Protocol tips
- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Publication protocol

MiRNAs reverse transcription and PCR reactions were performed on Trizol (Invitrogen, Carlsbad, CA, USA) isolated total RNAs using TaqMan MicroRNA Assays kit (Applied Biosystems, Foster City, CA, USA). Quantitative PCR on mRNAs was performed as previously described [16]. Western blots were done on total lysates or on nuclear/cytoplasmic fractions as previously described [16], with the following antibodies: anti-human TUBB3 polyclonal (1:1000, Covance, Princeton Township, NJ), anti-HuR (1:500, Santa Cruz, Santa Cruz, CA), anti-β-actin (1:5000, Sigma, Saint Louis, MO), anti-SNRP70 (1:1000, Abcam, Cambridge, UK), anti-GAPDH (1:5000, Abcam, Cambridge, UK). Blots were visualized by enhanced chemiluminescence procedures (Amersham, GE-Healthcare, Buckinghamshire, UK) as described by the manufacturers. The expression of HuR and TUBB3 was immunohistochemically assessed in a series of 220 ovarian cancers. Immunostaining for HuR was performed as previously described [16]. For the analysis of the expression of TUBB3, antigen retrieval procedure was performed by microwave oven heating in 10mM citric acid, pH 6.0 (2 times for 4 min.). TUBB3 protein was identified after overnight incubation at 4°C by using the monoclonal antihuman antibody (clone TUJ1;1:300; Covance) in 20% normal goat serum. The En Vision-mouse+ System-HRP (DAKO, Carpinteria, CA, USA) was used. Diaminobenzidine was used as a chromogen (DAB substrate System, DAKO). Sections were counterstained with haematoxylin.

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Manufacturer protocol

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