PAXgene Tissue RNA/miRNA Kit

Performance

Total RNA isolated with the PAXgene Tissue RNA/miRNA Kit is highly pure. Genomic DNA contamination is minimized and the purified RNA is ready to be used in downstream applications with no detectable inhibition of PCR. The resulting eluate includes smaller RNA molecules, such as 5.8S rRNA, 5S rRNA, tRNAs and miRNAs. Studies have shown that miRNA purified from PFPE tissue samples is reliably quantified, with high concordance to flash-frozen samples (Figure 1).

Principle

The PAXgene Tissue RNA/miRNA Kit enables purification of total RNA from tissues fixed and stabilized using the PAXgene Fixation and Stabilization products, which preserve tissue morphology and biomolecule integrity by avoiding destructive crosslinking and degradation found in formalin-fixed tissues. The purified RNA and miRNA have no inhibitory chemical modifications and thus, can be used in sensitive downstream applications (Figure 2). 

Procedure

PAxgene Tissue-fixed (PF) or PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissue samples are disrupted and homogenized in binding buffer. After centrifugation to remove cellular debris, optimal conditions are created for binding of RNA molecules to the silica membrane. Contaminants are then washed away and DNase I treatment removes any trace amounts of bound DNA. Total RNA, including miRNA, is then eluted in a low-salt elution buffer and denatured by heating (Figure 3).

The PAXgene Tissue RNA/miRNA Kit provides 2 protocols for purification of RNA, including miRNA, from different starting materials:

• Sections of PFPE tissues
• PF tissue samples (without paraffin embedding)

Applications

Total RNA and miRNA purified with the PAXgene Tissue RNA/miRNA Kit is ready to be used in a range of downstream research applications (see Resources), including:

• cDNA synthesis
• Gene expression arrays
• End-point RT-PCR
• Quantitative RT-PCR
• Detection and quantification of miRNA
• RNA sequencing