PAXgene Tissue RNA Kit

Total RNA purified using the PAXgene Tissue RNA Kit is of high quality and is highly pure (see figures "High-quality RNA from fixed tissue" and "RT-PCR of RNA purified from fixed tissue"). 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 200 nucleotides are purified. The procedure provides enrichment for mRNA since most RNAs <200 nucleotides (such as 5.8S rRNA, 5S rRNA, and tRNAs, which together comprise 15–20% of total RNA) are selectively excluded. The size distribution of the purified RNA is comparable to that obtained by centrifugation on a CsCl gradient, through which small RNAs do not sediment efficiently. For purification of small RNA, including microRNA (miRNA), we recommend using the PAXgene Tissue miRNA Kit.

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 cannot be removed completely, the resulting chemical modifications can cause inhibition in sensitive downstream applications such as quantitative PCR or RT-PCR.

The PAXgene Tissue System was developed  to enable both molecular and traditional pathology testing from the same specimen by stabilizing molecular content and preserving morphology. 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 total RNA, DNA, or miRNA. 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.

For purification of total RNA, the system requires the use of PAXgene Tissue Containers for tissue collection and stabilization, followed by RNA isolation and purification using the PAXgene Tissue RNA Kit. Together the container and kit provide a complete preanalytical solution for collection, fixation, and stabilization of tissue, and purification of high-quality RNA for molecular analysis.

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 from 3 to 7 days at room temperature or from 2 to 4 weeks at 2–8°C, depending on the type of tissue. Storage at –15°C to –30°C is also possible for at least 3 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 stabilized samples either before or after embedding in paraffin. See the PAXgene Tissue Container Product Circular for information about tissue fixation, stabilization, processing, and paraffin embedding.

The PAXgene Tissue RNA Kit provides 3 protocols for purification of total RNA from tissues fixed and stabilized in PAXgene Tissue Containers. Disruption and homogenization of the tissue sample is performed in the binding buffer, Buffer TR1. After a centrifugation step to remove residual cell debris, ethanol is added to the lysate to provide appropriate binding conditions for RNA. 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 is eluted in a low-salt elution buffer and denatured by heating (see figure "The PAXgene Tissue RNA procedure").

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

RT-PCR and quantitative, real-time RT-PCR

Expression-array and expression-chip analysis

cDNA synthesis

RNase and S1 nuclease protection

Northern, dot, and slot blot analysis

Primer extension