mirVana miRNA Isolation Kit - Thermo Fisher Scientific

1 PROTOCOLmirVana miRNA Isolation KitmiRNA Page 1 Tuesday, January 25, 2011 1:24 PMFor Research Use Only. Not intended for any animal or human therapeutic or diagnostic in this document is subject to change without BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. TO THE FULLEST EXTENT ALLOWED BY LAW, IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF, WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH USE LABEL LICENSE: RESEARCH USE ONLYThe purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser.

2 No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial services such as reporting the results of purchaser s activities for a fee or other form of consideration. For information on obtaining additional rights, please contact or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. 2011 Life Technologies Corporation. All rights Number 1560M Rev. C01/2011mirVana miRNA Isolation KitPart Numbers AM1560.

3 1A. BackgroundB. Summary of the mirVa n a m i R N A Is o l a t i o n K i t Pr o c e d u r eC. Reagents Provided with the Kit and StorageD. Materials Not Provided with the KitE. Related miRNA Isolation Kit Procedure.. 7A. Prepare the Wash SolutionsB. Equipment PreparationC. Sample Type and AmountD. Cell Lysis and Tissue DisruptionE. Organic ExtractionF. F i n a l R N A I s o l a t i o nG. Analyzing RNA after .. 16A. RNA Appears Degraded on GelB. RNA Yield Is Lower than Expected or InconsistentC. Contaminants in RNA; RNA Inhibits Enzymatic Procedures .. 19A. Isolation of Small RNAs from Total RNA SamplesB. Assessing RNA Concentration and Purity by UV AbsorbanceC. Gel Analysis of Small RNAsD. Detection of Small RNAs by Northern .. 26A. ReferencesB. Quality ControlC. Safety and Support.

4 29A. Obtaining SDSsB. Obtaining using this product, read and understand the Safety Information inthe appendix in this In the past few years, interest in the identification, detection, and use ofsmall RNA molecules has rapidly expanded. This interest is the result oftwo related lines of research. In one, small double-stranded RNAs (dsR-NAs) called small interfering RNAs (siRNAs) are used to silence theexpression of specific genes at the post-transcriptional level by a pathwayknown as RNA interference (RNAi). In the other, numerous small regu-latory RNA molecules, referred to as microRNAs (miRNAs), have beenshown to regulate target gene expression in various organisms. BothmiRNAs and siRNAs range between 15 30 nucleotides in length and arebiologically significant. Evidence is accumulating that these moleculesare related, and share common processing pathways (Zeng 2003).

5 The mirVana miRNA Isolation Kit was designed for purification ofRNA suitable for studies of both siRNA and miRNA in natural popula-tions. The kit employs an organic extraction followed by immobiliza-tion of RNA on glass-fiber filters to purify either total RNA, or RNAenriched for small species, from cells or tissue of the mirVana miRNA Isolation Kit ProcedureTraditional RNA Isolation methods are not well suited for Isolation of small RNAsVariations of two methods have historically been used to prepare RNAfrom natural sources ( tissue samples, whole organisms, cell cultures,bodily fluids): chemical extraction and immobilization on glass, oftenreferred to as solid-phase extraction. Chemical extraction methods usu-ally use highly concentrated chaotropic salts in conjunction with acidicphenol or phenol-chloroform solutions to inactivate RNases and purifyRNA from other biomolecules.

6 These methods provide very pure prep-arations of RNA; however, the RNA must typically be desalted and con-centrated with an alcohol precipitation step. Routine alcoholprecipitation does not quantitatively recover small nucleic acid mole-cules, making it ill-suited for the preparation of very small RNAs. The second method, solid-phase extraction, relies on high salt or saltand alcohol to decrease the affinity of RNA for water and increase itsaffinity for the solid support used. The use of glass (silica) as a solid sup-port has been shown to work for large RNAs in the presence of highmirVana miRNA Isolation Kit Summary of the mirVana miRNA Isolation Kit Procedure2concentrations of denaturing salts (Boom, et al. 1990). The conditionsroutinely used for solid-phase purification of RNA, however, do noteffectively recover small total RNA or RNA that is highly enriched for small RNAsThe mirVana miRNA Isolation procedure combines the advantages oforganic extraction and solid-phase extraction, while avoiding the disad-vantages of both.

7 High yields of ultra-pure, high quality, small RNAmolecules can be prepared in about 30 min (Figure1). Sample disruption and organic extractionThe first step of the mirVana miRNA Isolation Kit procedure is to dis-rupt samples in a denaturing lysis buffer. Next, samples are subjected toAcid-Phenol:Chloroform extraction which provides a robust front-endpurification that also removes most DNA (Chomczynski, 1987). Final RNA purification over glass-fiber filterAt this point there are separate procedures for purification of either totalRNA including very small RNA species or for purifying RNA that ishighly enriched for small RNA species and contains very little RNAlarger than about 200 1. Overview of the mirVana miRNA Isolation Kit Procedure. The sample is first lysed in a denaturing lysis solution which stabilizes RNA and inactivates RNases.

8 The lysate is thenextracted once with Acid-Phenol:Chloroform which removes most of the other cellular components, leaving a semi-pureRNA sample. This is further purified over a glass-fiber filter by one of two procedures to yield either total RNA or a sizefraction enriched in miRNAs. The glass-fiber filter procedure uses solutions formulated specifically for miRNA retentionto avoid the loss of small RNAs that is typically seen with glass-fiber filter 1, 2 & 3 Total RNAE luteWash 1, 2 & 3 Small RNAs (<200 nt)Add 1/3 volume ethanol to aqueous phase and passthrough first filterDisrupt sample inLysis Solution,add miRNAH omogenate AdditiveAcid Phenol:ChloroformextractFinal RNA IsolationCollect filtrateLysis andDisruptionExtractionSmall RNAsTotal RNAsOrganic~10 min~15 min~5 minAdd 2/3 volume ethanol to filtrateand pass through second filterAdd volumes ethanol and passthrough Summary of the mirVana miRNA Isolation Kit ProcedureIntroduction3 IMPORTANTTo isolate RNA for miRNAexpression profiling using miRNAarrays, we recommend following theprocedure for total RNA Isolation (not the enrichment procedure forsmall RNAs).

9 This makes it possibleto both quantitate the RNA and tocritically evaluate its quality, toverify that it is suitable for arrayanalysis. Then further purify themiRNA population ( , with theflashPAGE system) beforelabeling samples for miRNA purification of total RNA The procedure for Isolation of total RNA is similar to routine glass-fiberbinding procedures. Ethanol is added to samples, and they are passedthrough a Filter Cartridge containing a glass-fiber filter which immobi-lizes the RNA. The filter is then washed a few times, and finally theRNA is eluted with a low ionic-strength solution. Figures 2 and 3 onpage 3 compare RNA isolated using the mirVana miRNA Isolation Kitto RNA isolated using more traditional techniques. Final purification of RNA enriched for small RNAs To isolate RNA that is highly enriched for small RNA species,100% ethanol is added to bring the samples to 25% ethanol.

10 When thislysate/ethanol mixture is passed through a glass-fiber filter, large RNAsare immobilized, and the small RNA species are collected in the ethanol concentration of the filtrate is then increased to 55%, andit is passed through a second glass-fiber filter where the small RNAsbecome immobilized. This RNA is washed a few times, and eluted in alow ionic strength solution. Using this novel approach consisting of twosequential filtrations with different ethanol concentrations, an RNAfraction highly enriched in RNA species 200 nt can be 2. Comparison of miRNA Preps on Acrylamide Gel and Northern 3. Comparison of Total RNA Preps on Agarose Gel and Northern was prepared from two different tissues by doublephenol/guanidinium extraction (DPGE), glass-fiber filter(GFF), and mirVana miRNA Isolation Kit.