Description
General description
The High Pure RNA Tissue Kit is designed for the purification of total, intact RNA from tissue samples, free of any contaminating DNA. Low to medium throughput RNA isolation. 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. High pure RNA isolation kit can rapidly isolate intact total RNA from a broad range of research sample materials, including cultured cells, mammalian blood, white blood cells (WBCs), yeast, and bacteria. It rapidly isolates total RNA from mammalian tissues such as mouse liver, spleen, lung, and heart.
Capacity: The High Pure Spin Filter Tubes hold up to 700 μL sample volume.
Sample Material: Solid tissue (e.g., mouse liver, spleen, lung, heart): 1 - 25 mg.
Note: Sample size depends on the method used to prepare the tissue; larger samples (>10 mg) should be processed via rotor-stator homogenization.
Application
High Pure RNA Tissue Kit has been used:
to extract total RNA from human cells for cDNA library generation
to extract total RNA from cultured cells for real time PCR analysis
to extract total RNAs from rat hippocampi
to purify total RNA from cultured cells and human spinal cord
to isolate total RNA of tissues collected from ventricle walls
The isolated RNA can be used in many downstream applications:,
Northern blotting
RACE
primer extension
differential display
RNase protection assay
in vitro translation
Features and Benefits
Prepare RNA samples in approximately 30 minutes.
Obtain concentrated RNA that is suitable for downstream applications.
Minimize RNA loss with a kit that removes contaminants without precipitation or other handling steps that degrade RNA.
Eliminate the use of hazardous organic compounds such as cesium chloride, phenol, chloroform, and ethidium bromide.
Preparation Note
Tissue samples are disrupted and homogenized in the presence of a chaotropic salt (guanidine HCL) that inactivates RNases. The homogenate is then applied to the glass fiber fleece in a High Pure Spin Filter Tube. Under the buffer conditions used in the procedure, all nucleic acids bind to the glass fiber fleece, while contaminating substances (salts, proteins, and other cellular contaminants) do not. DNA in the preparation is digested with DNase I directly on the filter. Brief wash-and-spin steps readily remove the digested DNA fragments and other cellular contaminants. The remaining purified RNA is then eluted in a small volume of low-salt buffer.
Analysis Note
Tissue is disrupted and homogenized in Lysis/Binding Buffer and purified as described.
RNA yield is determined by measuring the optical density at 260 nm.
Integrity and size distribution are examined by the banding pattern of ribosomal RNA in a denaturing agarose gel.
10 μL of the RNA eluate is used in first strand synthesis with reverse transcriptase M-MuLV and p(dT)15 as primer. In the following PCR, accomplished using the Expand High Fidelity PCR System and specific primers for glycerinaldehyde 3-phosphate dehydrogenase (GAPDH), the expected amplification product of 983 bp is obtained.
Absence of contaminating DNA is examined by a PCR without a preceding RT reaction; no amplification product is obtained.
Other Notes
High Pure Technology and Silica Adsorption Kits
For life science research only. Not for use in diagnostic procedures.
Figure 1: Comparison of different homogenization procedures. Mouse liver was homogenized using various procedures. Total RNA was purified from each of these homogenates with the High Pure RNA Tissue Kit. Aliquots of the isolated RNA were reverse transcribed with primers specific for a region of the GADH mRNA. cDNA products were analyzed via gel electrophoresis.
Lane 1: Homogenization: Ultra Turrax; yield: 1.9 μg/mg tissue; A 260/ A 280 : 2.0
Lane 2: Homogenization: motor-driven, disposable plastic pestle; yield: 3 μg/mg tissue; A 260 /A 280 : 2.0
Lane 3: Homogenization: mortar and pestle, 20 G needle; yield: 1.5 μg/mg tissue; A 260 /A 280 : 2.0
Lane 4: Homogenization: manual disposable plastic pestle, 20 G needle; yield: 1.8 μg/mg tissue; A 260 /A 280 : 2.0
Lane 5: Homogenization: manual disposable plastic pestle; yield: 3.4 μg/mg tissue; A 260 /A 280 : 2.0
Lane 6: Homogenization: bead-vortex; yield: 3 μg/mg tissue; A 260 /A 280 : 2.0
Lane 7: Molecular weight marker
Lysis/Binding Buffer
DNase I, lyophilizate
DNase Incubation Buffer
Wash Buffer I
Wash Buffer II
Elution Buffer
High Pure Spin Filter Tubes (containing glass fiber fleece)
Collection Tubes
