miRNeasy Mini kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- 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
- 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

BEAS-2B cells exposed to 2.5 μg/cm2 of UFP or medium alone were collected at 1, 4, 8, 16, and 20 h, lysed, and stored in QIAzol Lysis reagent (Qiagen, Hilden, Germany) until RNA extraction. Three independent biological replicates were examined at each time point in UFP exposed and paired controls samples for a total of 45 samples. Total RNA was extracted using the miRNeasy extraction kit (Qiagen, Hilden, Germany) and eluted in RNase free-water, according to the manufacturer’s recommended guidelines. Quality and quantity of the total RNA samples were evaluated with 2100 Bioanalyzer (Agilent, Santa Clara, CA) and Nanodrop 1000 (Thermo Fisher Scientific, Wilmington, DE), respectively. Only RNA samples with RIN score more than 9 were considered. Same RNA samples were used either for RNA-seq and for qPCR validation. RNA-seq library was prepared from 1.5 μg of total RNA using TruSeq Stranded mRNA Library Prep Kit in paired end format (Illumina) following the manufacturer’s recommendations. RNA sequencing was carried out on Illumina HiSeq2500 platform (Illumina) at IGA technology service (Istituto Genomica Applicata, Udine, Italy). RNA-seq raw and processed data are deposited in the Array Express archive under the accession number E-MTAB-5157.

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Manufacturer protocol

Download the product protocol from Qiagen for miRNeasy Mini kit below.

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