- Fast – reliable purification of high-quality total RNA, genomic DNA and proteins in minutes
- Convenient – RNA, DNA and protein extracted from the same sample, with no splitting of the lysate, thereby improving yield and minimizing hands-on time
- Efficient - sequential extraction process ideal for maximizing recovery of RNA, DNA and protein from precious samples and small biopsies
- Versatile – optimized for recovery of all RNA molecules from large mRNA to small miRNA species, all DNA above 80 base pairs and total protein
- Safe - no hazardous phenol/chloroform extraction, CsCl centrifugation, LiCl or alcohol precipitation
The ISOLATE II RNA/DNA/Protein Kit provides a simple, efficient column-based method for the sequential isolation of total RNA, genomic DNA and proteins from a wide variety of starting materials, without the need for hazardous reagents such as phenol.
By combining the stringency of guanidine-thiocyanate lysis with the speed and ease-of-use of three silica-membrane purification columns, the ISOLATE II RNA/DNA/Protein Kit provides a fast method for the purification of high-quality total RNA, genomic DNA and proteins from a single sample of cultured cells, mammalian tissue, biofluids (including saliva, urine, semen and blood), bacteria, yeast, fungi or plants.
For applications that are sensitive to very small amounts of DNA, residual amounts of DNA remaining can be removed using a convenient on-column DNase treatment with RNase-free DNase I that is supplied with the kit.
The ISOLATE II RNA/DNA/Protein Kit allows for highly reliable integrative analysis of the transcriptome, genome and proteome, since the RNA, DNA and proteins are derived from the same sample and the same cell population.
The purified nucleic acids and proteins are of the highest purity and integrity and the ISOLATE II RNA/DNA/Protein Kit has been designed to deliver optimal performance in a wide variety of downstream applications including miRNA profiling using the EPIK Select Assays, novel biomarker discovery, siRNA gene silencing studies, gene expression profiling, as well as epigenetics, genotyping, transgenic analysis and cell line characterization.
Using the ISOLATE II RNA/DNA/Protein Kit means we would require less plates of cells, less steps in dividing up a sample (such as tissue) and most importantly time in sample preparation.
Michael Epis, The Harry Perkins Institute of Medical Research
Product SelectionPlease refer to the ISOLATE II Selection Chart to confirm the applications for which the ISOLATE II RNA/DNA/Protein Kit is recommended.
Certification of Analysis (COAs)
|ISOLATE II RNA/Protein Columns||50|
|ISOLATE II DNA Columns||50|
|Elution Tubes (1.7 mL)||150|
|Collection Tubes (2 mL)||150|
|Lysis Buffer TX||40 mL|
|Wash Buffer W1 (Concentrate)||2 x 38 mL|
|Wash Buffer W2 (Concentrate)||15 mL|
|Wash Buffer W3||30 mL|
|DNase I Reaction Buffer DRB||6 mL|
|DNase I Solution (RNase-free)||0.8 mL|
|RNA Elution Buffer||6 mL|
|DNA Elution Buffer||15 mL|
|Protein Elution Buffer||8 mL|
|Protein Binding Buffer||8 mL|
|Protein Neutralization Buffer||4 mL|
|Protein Loading Dye||2 mL|
|Bench Protocol Sheet||1|
Storage & Stability
The DNase I Solution should be stored at -20°C upon arrival. All other components should be stored dry and at room temperature.
When stored under the recommended conditions and handled correctly, full activity of reagents is retained until the expiry date indicated on the outer box label.
An increase of A230 values may be caused by different substances, like carbohydrates, peptides and phenol. A bad A230/A260 ratio in RNA samples is mostly due to a contamination with guanidinium thiocyanate which is present in several reagents used for RNA extraction, for instance in the lysis buffer. In contrast to a bad A280/A260 ratio it does not automatically reflect a bad RNA quality. Currently there is no consensus about a lower limit of this ratio and mostly a carry-over of guanidinium thiocyanate does not affect the reliability of downstream applications. Nevertheless, an extra washing step with RW2 would be helpful to avoid this problem. And it would be helpful to pipet the flow-through out of the collection tube, instead of pouring it off.
Agarose gel analysis of RNA samples can be both valuable and misleading. The pattern of bands on the gel can not only be indicative of the quality of the sample, but equally it can also only indicate that the gel tank, buffer or agarose is contaminated with RNase. A safer measure is to use a Bioanalyzer and look at the RIN value (RNA Integrity Number), which should be as close to 10 as possible, indicating that the RNA is not degraded. A spectrophotometer (ideally a microfluidic one) can also be used to determine the ratio of A260 to A280 for purity determination.
All these methods give an idea of the quality of total RNA which tends to be dominated with rRNA and tRNA. It is not uncommon for an agarose gel to show an abundance of 18S and 23S rRNA but on analysis for there to be little of the transcript of interest. Ideally RNA quality control should include an assessment of the presence of common transcripts (from reference genes, such as GAPDH) using RT-PCR or RT-qPCR.
If the yield of RNA is low, it is best to first check that all the solutions used and the equipment employed is largely free of RNase. Solutions should be prepared with the highest quality reagents available, preferably ones that have a certificate of analysis to show that they are free of DNase and RNase. If there is confidence that RNase contamination is at a minimum, then it may be worth ensuring that the sample is properly homogenised before lysis and to check a sample of the lysed material under a light microscope to ensure that all the cells are disrupted.
RNA samples that have been contaminated with RNase (either during processing or those that have been sent from another lab) can be purified using the ISOLATE II RNA Micro Kit.
For the optimal recovery of large RNA/DNA fragments it is necessary to optimize the elution step. To increase the recovery rate it would be helpful to use a larger volume for elution, incubate the column with the elution buffer prior to centrifugation and use repeated elution steps. To avoid excessive dilution it may be helpful to reapply the eluted fraction again. Furthermore it could be helpful for DNA elution to heat the elution buffer to 70°C.