1
|
<p>Total RNA was extracted using Trizol<sup>®</sup> reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. Approximately 30 mg of PFC tissue was added into a 2 ml tube containing 700ul QIAzol Lysis Reagent and one 5 mm stainless steel bead (Qiagen, Hilden, Germany). The tissue was homogenized for 2 minutes in a Tissue Lyser II (Qiagen, Hilden, Germany) with a speed frequency of 30 r followed by incubating for 5 minutes.140 <a name="_Hlk32821176">µ</a>l chloroform was added into the homogenate, shaken vigorously for 15 seconds, and centrifuged for 15 minutes at 12,000 x <em>g</em> at 4 ℃. 280 µl upper aqueous was then transferred into a new collection tube containing 500 µl 100% ethanol. The mixture was loaded into a RNeasy miniâ€spin column (Qiagen, Valencia, CA, USA), once with Buffer RWT and twice with Buffer RPE purification. All RNA had been treated with DNase to avoid DNA contamination, and verified by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA with OD260/280 > 1.8 and RIN > 8.0 were used for library preparation</p>
|
