The ISOLATE II miRNA Kit provides a simple, efficient column-based method for the isolation of miRNA and total RNA from a wide variety of starting materials, without the need for hazardous reagents such as phenol, which has been reported to introduce significant bias in recovery of selected small RNAs.
By combining the stringency of guanidinium-thiocyanate lysis with the speed and ease-of-use of two silica-membrane purification columns, the ISOLATE II miRNA Kit provides a fast method for the purification of high-quality total RNA (on the first column) and miRNA (on the second column) from cultured animal cells, small tissue samples, bacterial cells, plants and blood.
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 miRNA Kit has been designed to deliver optimal performance in miRNA expression profiling and quantification using qPCR, microarrays, small RNA library construction for small RNA-Seq, as well as single cell assays.
|ISOLATE II Large RNA Removal Columns||25|
|ISOLATE II miRNA Columns||25|
|Collection Tubes (2 mL)||50|
|Elution Tubes (1.7 mL)||50|
|Lysis Buffer RX||40 mL|
|Wash Buffer W1||38 mL|
|DNase I Solution (RNase-free)||0.8 mL|
|DNase I Reaction Buffer DRB||6 mL|
|RNA Elution Buffer||6 mL|
|Bench Protocol Sheet||1|
The DNase I 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.
We recommend our ISOLATE II Biofluids RNA, miRNA and Plant miRNA kits. The miRNA kits are able to capture all small RNA species (<200 nt) from plant cells and tissues and to purify high quality large RNA (>200 nt) from the same sample in parallel. The ISOLATE II miRNA Kits have been developed to overcome the bias observed with small RNA isolation using phenol-based techniques, due to its proprietary small RNA separation and enrichment technology, as well as highly optimized chemistry. The Biofluids RNA kit isolates all sizes of RNA from large mRNA, viral RNA and ribosomal RNA down to small RNAs such as miRNA and siRNA. The ISOLATE II Biofluids RNA Kit is compatible with very limited, or challenging sample sources containing low RNA content.
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.