RNeasy FFPE Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- To allow complete penetration by formalin, use tissue samples less than 5 mm thick	- Perform all centrifugation steps using a microcentrifuge placed at 15–25°C	- Do not exceed fixation time of 24 hours as it results in poor performance in downstream assay

Upstream tips
- To allow complete penetration by formalin, use tissue samples less than 5 mm thick
Protocol tips
- Perform all centrifugation steps using a microcentrifuge placed at 15–25°C
Downstream tips
- Do not exceed fixation time of 24 hours as it results in poor performance in downstream assay

Publication protocol

When using paraffin embedded formalin tissue sections Qiagen RNeasy FFPE kit (Qiagen, Valencia, CA) was used according to the manufacturer's instructions to extract mRNA. For mRNA extraction for fresh tissues, specimens were placed into Tri-Reagent (Fisher Scientific) supplemented with glassbeads (1.0 mm diameter, BioSpec Products, Inc., Bartlesville, OK) immediately after collection and processed as recommended by the manufacturer. First-strand cDNA conversion was performed using iScript and real time PCR was conducted with SYBR Green according to manufacturers’ protocols (Bio-Rad Laboratories, Hercules, CA). The gene coding for the ribosomal protein RPLP0 was used as reference. Gene accession numbers were as follows: NM_001002.3 (RPLP0), NM_003101 (SOAT1), NM_004345 (LL37), NM_004942.2 (HBD2), and NM_0011081551.2 (HBD3). Primers had been designed by IDT (LL37 and SOAT1, Integrated DNA Technologies, Inc., San Diego, CA) or using NCBI Primer blast (RPLP0, HBD2, and HBD3). All PCR primers were purchased from IDT. Amplicons had been previously sequenced (DNA Core Facility, Beckman Research Institute, City of Hope, Duarte, CA, or Retrogen Inc., San Diego, CA) and were computer matched with the known cDNA sequences of each molecule.

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Manufacturer protocol

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

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