High Pure RNA Isolation Kit
Low to medium throuhgput, mini scale RNA isolation.
For life science research only. Not for use in diagnostic procedures.
The High Pure RNA Isolation Kit isolates total RNA from cultured cells, free of any contaminating DNA. Other sample materials, such as whole blood, yeast, and bacteria require a pre-lysis treatment.
- Quickly process multiple samples. A single sample preparation is completed in 25 minutes, and multiple samples can be prepared in 45 minutes.
- Prevent RNA loss. Precipitation or solvent extraction steps are not required.
- Avoid DNA contamination. Integrated DNA digestion and DNase removal eliminates interference from genomic DNA (see Figure 1).
- Obtain concentrated RNA. RNA is eluted in a 50 μL volume.
- Obtain high sensitivity, reproducibility, and specificity for downstream applications.
- Lysis/Binding Buffer
- DNase I, recombinant, lyophilizate
- DNase Incubation Buffer
- Wash Buffer I
- Wash Buffer II
- Elution Buffer
- High Pure Filter Tubes
- Collection Tubes
The High Pure RNA Isolation Kit rapidly isolates small amounts of total RNA from a variety of samples, such as cultured cells, whole blood, yeast, and bacteria. The isolated RNA can be used in many downstream applications:
- Northern blotting (see Figure 2)
- Primer extension
- Differential display
- cDNA library construction
- RNase protection assays
- In vitro translation
Figure 1: Effect of DNase treatment on mammalian RT-PCR templates prepared with the High Pure RNA Isolation Kit. RNA was isolated from six identical samples, each containing 106 K-562 human lymphocyte cells. Four samples were treated with DNase as described in the Instructions for Use. For two samples, the DNase step was omitted. The isolated RNAs were used as template in a two-step RT-PCR.
Left Panel: Amplification of a fragment of the GAPDH gene.
Right Panel: In a control reaction, amplification was performed without prior first strand cDNA synthesis.
Result: After first strand cDNA synthesis, the expected fragment could be amplified from all samples (left panel). However, in the samples that were not treated with DNase, a fragment was also amplified in the control (no cDNA synthesis) reactions, indicating that those samples contained residual genomic DNA (right panel; lanes 6 and 7).
Figure 2: Northern blot analysis of RNA isolated with the High Pure RNA Isolation Kit and a competitor kit. RNA was isolated from 106 K-562 human lymphocyte cells with each kit, and yield was determined spectrophotometrically. The indicated amounts of RNA from each isolate were separated on a denaturing formaldehyde gel and subjected to northern blotting. Actin RNA was detected with a specific DIG-labeled RNA probe.
Result: RNA isolated with the High Pure RNA Isolation Kit produced stronger signals and lower background hybridization.
Nucleic acids bind to the surface of the glass fiber 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 700 μL sample volume.
Typical RNA yields from different samples obtained with the High Pure RNA Isolation Kit :
|Starting Material||Sample Size||Time Required||Average RNA Yield|
|106 cells||30 min||15 μg|
|Whole blood, human||200 µL||50 min, including RBC lysis||Sufficient for 10 RT-PCR reactions|
(S. cerevisiae )
|108 cells||50 min, including lyticase digest||20 μg|
Gram negative bacteria
|109 cells||90 min, including lysozyme digest||50 μg|
Gram positive bacteria
(B. subtilis )
|109 cells||90 min, including lysozyme digest||35 μg|
A single reagent which contains a chaotropic salt and detergent lyses the sample and simultaneously inactivates RNases. After cell debris is removed by centrifugation, the lysate is applied to the glass fiber fleece in a High Pure Spin Filter Tube. Under the buffer conditions used in the procedure, 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 contaminating substances. Once purified, the RNA can be easily eluted in a small volume of low-salt buffer.
1 x 106 K-562 cells are treated as described in the protocol for cultured cells. RNA yield is determined by measuring the optical density at 260 nm. At least 10 µg of total RNA are isolated. Integrity and size distribution are examined by the banding pattern of ribosomal RNA in a denaturing agarose gel. 100 ng of isolated total RNA is used in first strand synthesis with reverse transcriptase M-MuLV and p(dT)15 as a primer. In the following PCR, accomplished using the Expand High Fidelity PCR System and specific primers for glyceraldehyde 3-phosphate dehydrogenase (G3PDH), the expected amplification product of 983 bp is obtained. Absence of contaminating DNA is examined via PCR without a preceding RT reaction; no amplification product is obtained. All kit components are function tested for absence of RNases.