By combining the stringency of guanidinium-thiocyanate lysis with the speed and ease-of-use of silica-membrane purification, the ISOLATE II Biofluid RNA Kit provides a fast method for the purification of high-quality total RNA from biofluids including blood, serum, plasma, urine, saliva and cerebrospinal fluid.
A genomic DNA column is used to remove contaminating genomic DNA. For applications that are very sensitive residual amounts of DNA remaining can be removed using DNase treatment with RNase-free DNase I that is supplied with the kit.
Viral RNA can also be isolated from biofluid sources, as well as from cultured cells and tissues. ISOLATE II Biofluids RNA Kit isolates all sizes of RNA from large mRNA, viral RNA and ribosomal RNA (rRNA) down to small RNAs such as microRNA (miRNA) and short interfering RNA (siRNA).
ISOLATE II Biofluids RNA Kit has been designed to deliver optimal performance in RT-qPCR in conjunction with either the SensiFAST cDNA Synthesis Kit and SensiFAST Real-Time PCR Kits, or the SensiFAST One-Step Real-Time RT-PCR Kits. Additionally, the ISOLATE II Biofluid RNA Kit can be used to purify samples prior to RT-PCR amplification using the Tetro cDNA Synthesis Kit and Bioline PCR portfolio MyTaq DNA Polymerase.
|ISOLATE II RNA Columns||50|
|ISOLATE II Genomic DNA Removal Columns||50|
|Collection Tubes (2 mL)||100|
|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 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.
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.
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.