PAXgene Tissue RNA/miRNA Kit

Tissue samples fixed and stored in PAXgene Tissue Containers can be paraffin-embedded and used for pathological studies as well as for subsequent purification of miRNA, RNA, and/or DNA. The PAXgene Tissue miRNA Kit provides purification of total RNA, including RNA from approximately 18 nucleotides, from tissues fixed and stabilized in PAXgene Tissue Containers. Purification is carried out using silica-based RNA purification technology in a spin-column format. Used with the containers, the kit provides a complete preanalytical solution for collection, fixation, and stabilization through to purification of high-quality miRNA and total RNA for molecular analysis.

Together the container and kit provide a complete preanalytical solution for collection, fixation, and stabilization of tissue, and for purification of high-quality RNA, including miRNA, for molecular analysis (see figure " Efficient purification of miRNA from fixed tissue stored in PAXgene Tissue Containers").

Total RNA purified using the PAXgene Tissue miRNA Kit is highly pure. Genomic DNA contamination is minimized, and purified RNA is ready to use in downstream applications with no detectable PCR inhibition. All RNA molecules longer than 18 nucleotides are purified.

Current tissue fixation methods used in traditional histology are of limited use for molecular analysis. Fixatives that contain formaldehyde cross-link biomolecules and modify nucleic acids and proteins. During tissue fixation, storage, and processing, cross-links lead to degradation of nucleic acids. Since cross-links can not be removed completely, the resulting chemical modifications can cause inhibition in sensitive downstream applications such as quantitative PCR or RT-PCR. In order to enable both molecular and traditional pathology testing from the same specimen, a method is needed for stabilization of molecular content and preservation of morphology.

PreAnalytiX has developed the PAXgene Tissue System to meet such needs. The system consists of a tissue collection device (the PAXgene Tissue Container for collection, stabilization, storage, and transportation of human tissue specimens) and kits for purification of miRNA, total RNA, or DNA. PAXgene Tissue Containers provide tissue fixation for histopathology studies and enable purification of high-quality nucleic acids from the same sample for molecular analysis. The fixation and stabilization method preserves tissue morphology and the integrity of nucleic acids without destructive cross-linking and degradation found in formalin-fixed tissues.

PAXgene Tissue Containers are dual-chamber containers prefilled with 2 reagents. PAXgene Tissue Fix rapidly penetrates and fixes the tissue. After fixation, the tissue is removed from the PAXgene Tissue Fix and transferred to PAXgene Tissue Stabilizer in the second chamber of the same container. When the tissue is stored in PAXgene Tissue Stabilizer, nucleic acids and morphology of the tissue sample are stable for a minimum of 3 and a maximum of 7 days at room temperature or for up to 8 weeks at 2–8°C, depending on the type of tissue. Storage at –15 to –30°C is also possible for at least 26 months without any negative effects on the morphology of the tissue or the integrity of the nucleic acids.

Stabilized samples can be embedded in paraffin for histological studies. Nucleic acids can be isolated from the PAXgene Tissue fixed, paraffin-embedded (PFPE) samples either before or after embedding in paraffin.

The PAXgene Tissue miRNA Kit provides 3 protocols for purification of total RNA from tissue fixed and stabilized in PAXgene Tissue Containers, including RNA molecules smaller than 200 nucleotides, such as 5.8S rRNA, 5S rRNA, tRNAs, and miRNAs. Optimized binding and washing conditions ensure the purification of RNA molecules as small as 18 nucleotides. As a prerequisite, the tissue must be fixed and stabilized in PAXgene Tissue Containers.

Disruption and homogenization of the tissue sample is performed in the binding buffer, Buffer TM1. After a centrifugation step to remove residual cell debris, ethanol is added to the lysate to provide appropriate binding conditions for all RNA molecules 18 nucleotides and longer. The sample is then applied to a PAXgene RNA MinElute spin column where the total RNA binds to the membrane and contaminants are efficiently washed away. Between the first and second wash steps, the membrane is treated with DNase I to remove trace amounts of bound DNA. After the wash steps, RNA including miRNA is eluted in a low-salt elution buffer and denatured by heating (see figure " The PAXgene Tissue miRNA procedure").

Specifications for fixation and storage conditions in PAXgene Tissue Fix and PAXgene Tissue Stabilizer were determined using animal tissues.

The purified miRNA and total RNA is ready to use in a wide range of downstream applications, including:

• Northern blot analysis
• RT-PCR and quantitative, real-time RT-PCR
• Microarray analysis