Total RNA Purification Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- For lysis/cell disruption check the protocol beforehand to see which reagents/lab materials are needed		- Minimal DNA is left over using this kit, however if DNA is undesired an additional RNase free DNase treatment is warranted.

Upstream tips
- For lysis/cell disruption check the protocol beforehand to see which reagents/lab materials are needed
Downstream tips
- Minimal DNA is left over using this kit, however if DNA is undesired an additional RNase free DNase treatment is warranted.

Publication protocol

Total RNA was isolated using the following kits: Isolate II RNA mini kit (Bioline, London, UK), miRVana microRNA isolation kit (Thermo Fisher Scientific, Waltham, MA, USA), Total RNA isolation kit (Norgen Biotek, Thorold, ON, Canada), miRNeasy mini kit (Qiagen, Hilden, Germany), and TRIzol Reagent (Thermo Fisher Scientific). Frozen tissue samples were weighed and processed on ice to prevent thawing. In preparation for RNA extraction 5–10 mg of tissue (weights differed by tissue but were consistent for all kits) was added to 700 μl of the appropriate lysis buffer. Samples were homogenised using a TissueLyser II (Qiagen) with 5 mm stainless steel beads for 3 × 1 min cycles at 30 Hz, with resting on ice in between. Homogenates were subjected to a single freeze/thaw cycle to aid cell lysis and then cleared by centrifugation at 10,000 x g for 5 min at 4 °C. RNA extractions were performed according to manufacturer’s instructions, with the exception of an additional chloroform extraction step for the TRIzol reagent protocol as illustrated in Fig. 2. RNA was eluted in 35 μl of RNase free water and stored at − 80 °C. Note that for all methods the total RNA extraction method was followed. miRNA enrichment was not performed for any method as efficient isolation of both miRNA and mRNA transcripts from the same sample is of interest.

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Manufacturer protocol

Download the product protocol from Norgen Biotek for Total RNA Purification Kit below.

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