Description
General description
Low to medium throughput RNA isolation from FFPE sample materials.
Application
The High Pure RNA Paraffin Kit isolates total RNA from paraffin embedded, fresh-frozen tissue. The quality of RNA obtained is suitable for relative quantification of mRNA with RT-PCR, especially on the Lightcycler® Carousel-Based System. Additional applications include:
RT-PCR
Differential display RT-PCR
cDNA synthesis
Primer extension
Features and Benefits
The High Pure RNA Paraffin Kit isolates total RNA from formalin-fixed, paraffin-embedded tissue as well as from fresh-frozen tissue-research samples for direct use in RT-PCR.
Isolate RNA suitable for RT-PCR from archived paraffin tissue (up to 15 years old).
Obtain a concentrated product that is ready-to-use (no RNA precipitation required).
Quickly prepare total RNA from tissue sections in approximately 2 hours (after an overnight digestion).
Obtain highly pure RNA for use in relative mRNA quantification procedures (e.g ., with the Lightcycler® System).
Eliminate the use of hazardous organic compounds such as cesium chloride, phenol, chloroform, and ethidium bromide.
Preparation Note
Tissue samples are disrupted and homogenized during an overnight incubation with Proteinase K (paraffin samples) or by using a suitable tissue homogenizer (fresh-frozen tissue). Nucleic acids bind in the presence of a chaotropic salt specifically to the surface of the glass fibers pre-packed in the High Pure Purification Filter Tube (1). The binding reaction occurs within seconds due to the disruption of the organized structure of water molecules and the interaction with nucleic acids. The binding process is specific for nucleic acids in general, but the binding conditions are optimized for RNA. Bound RNA is purified in a series of brief wash-and-spin steps to remove cellular components. After elution from the column, residual DNA is digested by incubating the eluate with DNase I. A second incubation step with Proteinase K improves the purity of RNA. Finally, a low-salt elution releases the RNA from the glass fiber fleece.
Analysis Note
Formalin-fixed, paraffin-embedded tissue sections are homogenized by overnight Proteinase K digestion and purified as described. RNA yield is determined by measuring the optical density at 260 nm. The RNA eluate is used in one-step RT-PCR with specific primers for the β2M-gene. In the following LightCycler® PCR, using the LightCycler® RNA Amplification Kit SYBR Green I and specific primers for β2M, the expected amplification signal is obtained at a cp-value less then 24. Absence of contaminating genomic DNA is examined by a LightCycler® PCR without a reverse transcriptase step; no amplification product is obtained.
Nucleic acids bind to the surface of the glass fleece in the presence of a chaotropic salt (guanidine HCl). This allows the High Pure Filter Tube to specifically immobilize nucleic acids (both DNA and RNA) while they are freed of contaminants. For RNA isolation, the binding conditions can be optimized to ensure immobilization of all the RNA.
Capacity: The High Pure Spin Filter Tubes hold up to 800 μL sample volume.
Sample Material:
5 - 10 μm sections from formalin-fixed, paraffin-embedded tissue (e.g., colon, breast, liver, kidney, spleen of mammalian species, including human-research samples).
20 – 30 mg fresh-frozen solid tissue.
3 × 5 μm tissue sections from fresh-frozen tissue.
Other Notes
High Pure Technology and Silica Adsorption Kits
Tissue Lysis Buffer
Proteinase K, PCR-grade
Binding Buffer
Wash Buffer I
Wash Buffer II
DNase I
DNase Incubation Buffer
Elution Buffer
High Pure Filter Tubes
Collection Tubes
For life science research only. Not for use in diagnostic procedures.
Purified RNA is free of DNA, nucleases, and all cellular and sample components that interfere with RT-PCR. Typically, RNA fragments isolated from formalin-fixed tissue ranged from 150 up to 1500 bases. The bulk of RNA is 250 bases in length.
Legal Information
LightCycler is a registered trademark of Roche
