RNeasy FFPE Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	All FFPE sections were deparaffinised using an automated protocol on the Leica autostainer XL, involving immersion into xylene twice followed by immersion into 100% ethanol twice then left to air dry for 15 min, with the exception of the two Promega kits where no deparaffinisation was performed. All extraction kits were used according to the manufacturer’s instructions including elution volumes quoted and all optional DNase 1 steps in RNA extractions.

Protocol tips

All FFPE sections were deparaffinised using an automated protocol on the Leica autostainer XL, involving immersion into xylene twice followed by immersion into 100% ethanol twice then left to air dry for 15 min, with the exception of the two Promega kits where no deparaffinisation was performed. All extraction kits were used according to the manufacturer’s instructions including elution volumes quoted and all optional DNase 1 steps in RNA extractions.

Publication protocol

A total of 30 samples from 6 FFPE cell line blocks were generated using each extraction method yielding a total of 240 samples as one method extracted both DNA and RNA. RNA extractions were performed on the following cell line blocks: KCL22, MDA-MB-468, MDA-MB-453 (fixed less than 6 months prior to this work) and MDA-MB-231, MDA-MB-468 and MDA-MB-453 (fixed 2–3 years prior to this work). DNA extractions were performed on the following cell line blocks T47D, MDA-MB-468 and MDA-MB-453 from each age group (Table 2). 15 consecutive 5 μm sections were generated from each block and processed as 5 μm, 10 μm (2 combined sections), 15 μm (3 combined sections), 20 μm (4 combined sections) and 25 μm (5 combined sections) in each extraction method. All FFPE sections were deparaffinised using an automated protocol on the Leica autostainer XL, involving immersion into xylene twice followed by immersion into 100% ethanol twice then left to air dry for 15 min, with the exception of the two Promega kits where no deparaffinisation was performed. All extraction kits were used according to the manufacturer’s instructions including elution volumes quoted and all optional DNase 1 steps in RNA extractions. Following deparaffinisation, the surface area of the cell line pellet was checked by eye from consecutive sections within a block to ensure they matched. All RNA samples were stored at −80 °C and all DNA and cDNA samples were stored at −20 °C throughout the study.

Out of the four RNA extraction kits evaluated the RNeasy FFPE kit, from Qiagen, gave superior geometric mean yields, whilst the Maxwell 16 automated method, from Promega, yielded the highest quality RNA by quantitative real time RT-PCR. Of the DNA extraction kits evaluated the PicoPure DNA kit, from Life Technologies, isolated 2–14× more DNA. A miniaturised qPCR assay was developed for DNA quantification and quality assessment.

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Manufacturer protocol

Download the product protocol from Qiagen for RNeasy FFPE Kit below.

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