QIAzol Lysis Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- To promote dissociation of nucleoprotein complexes, place the sample at room temperature (15–25°C) for 5 min after homogenization		- Recommends for a cleanup of the redissolved RNA using RNeasy® Kits, which are based on silica-membrane technology, in order to remove any contaminating phenol - If A260/A280 is low or if DNA containation is high, reduce the amount of starting material and/or increase the volume of QIAzol Lysis Reagent and the homogenization time

Upstream tips
- To promote dissociation of nucleoprotein complexes, place the sample at room temperature (15–25°C) for 5 min after homogenization
Downstream tips
- Recommends for a cleanup of the redissolved RNA using RNeasy® Kits, which are based on silica-membrane technology, in order to remove any contaminating phenol - If A260/A280 is low or if DNA containation is high, reduce the amount of starting material and/or increase the volume of QIAzol Lysis Reagent and the homogenization time

Publication protocol

In a first step, four rat PCLS per tube were used for RNA isolation. Several commercially available kits were used for this according to the suppliers’ instructions: RNeasy Mini Kit (Qiagen, Hilden, Germany), QIAzol® (Qiagen, Hilden, Germany), MagJET (Thermo Fisher Scientific, Dreieich, Germany), and MagMAX™ (Ambion™/Thermo Fisher Scientific, Dreieich, Germany). Disruption and homogenization of PCLS in the respective solutions was performed using an Ultra-Turrax® (T8, IKA, Stauffen, Germany).

In a second step, optimized RNA extraction was achieved as follows: two PCLS were pooled, followed by disruption and homogenization of PCLS in 400 µL RLT lysis buffer (Qiagen, Hilden, Germany) using an Ultra-Turrax®. The homogenate was transferred to 1 volume of phenol/chloroform, carefully shaken for 30 s, and centrifuged for 5 min at 12,000×g. Subsequently, 1 volume of chloroform/isoamyl alcohol was added, again carefully shaken for 30 s, and centrifuged for 5 min at 12,000×g. The aqueous phase was transferred and RNA was cleaned up with MagMAX™ magnetic beads including the spin procedure step according to the supplier’s instructions. Total RNA was dissolved in RNase-free water and stored at −80 °C. For some samples an additional clean up step was performed using the RNeasy Mini Kit clean up protocol (Qiagen, Hilden, Germany).

RNA from A459 cells was isolated with the commercially available RNeasy Mini Kit according to the supplier’s instructions.

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Manufacturer protocol

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