Navigation Links
Got Small RNA?

Ensure the Presence of Small RNA, Including miRNAs, in Your RNA Sample

Isolate both large and small RNA efficiently
Enrich for small RNA (10-200 nt)
Purify RNA quickly (~30 minutes)


Not All RNA Isolation Procedures Purify Small RNAs

Ambion scientists have developed a line of mirVana RNA Isolation Kits for the preparation of miRNA and other small RNA species. Other RNA purification procedures rely on organic extraction followed by alcohol precipitation or adsorption of nucleic acids on a silicate matrix, such as a glass fiber filter (GFF). Unfortunately, alcohol precipitation is time consuming and sometimes inefficient at recovering small RNAs. Furthermore, adsorption to solid supports (e.g. RNeasy Kit from Qiagen) usually results in loss of most of the small RNA fraction, including small ribosomal RNA (5S rRNA), transfer RNA (tRNA), microRNA (miRNA), or small interfering RNA (siRNA).


Efficient Recovery of Small RNAs

The mirVana miRNA Isolation Kits, which employ organic extraction followed by purification on a GFF, use specialized binding and wash solutions that are optimized to prevent loss of the small RNA fraction. The fast and efficient GFF-based method isolates total RNA ranging in size from kilobases down to 10-mers. Figure 1 demonstrates that RNA isolated by the mirVana miRNA Isolation Kit or the mirVana PARIS Kit retains the small RNA fraction.

Figure 1. Differential Recovery of Small RNAs. Total RNA was isolated from 1x106 HeLa cells with the indicated kit as per protocol. Total RNA (1 g) was resolved on a 15% denaturing acrylamide gel and stained with ethidium bromide or analyzed by solution hybridization assay with the mirVana miRNA Detection Kit (Ambion) and a probe specific for miR-16 prepared by in vitro transcription with the mirVana Probe Construction Kit (Ambion). The gel was exposed for 6 h at -80C.


You Can Also Enrich the Small RNA Fraction

The mirVana Isolation Kit also provides reagents and a procedure to enrich the population of RNAs that are 200 bases and smaller. Small RNA enrichment results in lower background and enhanced sensitivity of small RNA detection by solution hybridization, Northern analysis, and other methods, as compared to the same assay using total RNA. A representative enrichment/depletion example with the mirVana PARIS Kit is presented in Figure 2. The depleted and enriched fractions, or total RNA, were purified from the same number of HeLa cells. Analysis of each fraction on a denaturing agarose gel, a denaturing acrylamide gel or the Agilent 2100 bioanalyzer showed that 28S, 18S, and 5.8 rRNAs were quantitatively recovered in the fraction depleted in small RNA. In contrast, RNAs smaller than 200 nt such as 5S RNA and tRNA were significantly enriched in the complementary "enriched" fraction when compared to the total RNA sample.

Figure 2. Small RNA Enrichment Procedure. Total RNA or fractions depleted or enriched in small RNA were isolated from 1x106 HeLa cells with the mirVana PARIS Kit as per protocol. A 1 g aliquot of each fraction was resolved on a 1.2% denaturing glyoxal agarose gel or a 15% denaturing acrylamide gel and stained with ethidium bromide. The same fractions were also analyzed with an RNA 6000 NanoChip on the Agilent 2100 bioanalyzer.


One Sample mRNA +Small RNA + Protein

In addition to efficient isolation of RNA of all sizes and the optional small RNA enrichment procedure, the mirVana PARIS Kit was also designed to quantitatively recover total proteins from the same sample. This is especially important in experiments where the expression level of both the active miRNA or siRNA and the corresponding target protein need to be measured. Figure 3 demonstrates the effectiveness of the mirVana PARIS Kit for preparing samples for this type of experiment. Here, GAPDH knockdown was triggered by electroporation of a GAPDH-specific siRNA in a primary human cell line (normal human umbilical vein endothelial cells or HUVEC). A reduction of GAPDH expression was observed both at the mRNA and protein levels in these cells, but not in HUVEC cells electroporated with a control Negative Control #1 siRNA sequence (NC). No variation of expression levels was detected for control mRNA (-actin), small RNA (miR-16), and protein (Ku p70).

Figure 3. Using the mirVana PARIS Kit to Analyze RNAi. HUVEC cells (1.5x106) were electroporated with 10 g of an siRNA targeting GAPDH mRNA or the Silencer Negative Control #1 siRNA (NC, Ambion) in 400 ml of siRNA Electroporation Buffer (Ambion). Total RNA and protein were isolated with the mirVana PARIS Kit 48 hours after electoporation. Total RNA (1 g) was used to detect the indicated RNA species by Northern blot or by solution hybridization assay with the mirVana miRNA Detection Kit (Ambion). RNA probes were prepared by in vitro transcription (mRNA probes: MAXIscript Kit, Ambion) or by 5' end labeling (miRNA and siRNA probes: mirVana Probe & Marker Kit, Ambion). Western blots were performed with 15 g of total protein and antibodies specific for GAPDH or Ku p70 proteins (Ambion).


Complete Kits

The mirVana Isolation Kits include sufficient reagents to perform 40 total RNA isolations or 20 enrichment/depletion procedures. Each purification can accommodate 100-107 cells or 1-100 mg of tissue for the mirVana PARIS Kit, up to 1 g of tissue for the mirVana miRNA Isolation Kit.


Scientific Contributors
Lesslie Beauchamp, Dmitriy Ovcharenko, Emmanuel Labourier Ambion, Inc.


back to top


Ordering Information
Cat# Product Name Size 1556 mirVana PARIS Kit up to 40 purifications 1560 mirVana miRNA Isolation Kit up to 40 purifications
'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Fast Isolation of Total RNA from Small Numbers of Cells
2. Detecting Attomole Amounts of Small RNA
3. New Tools for Small RNA Analysis
4. Rapidly Prepare Probes and Markers for Small RNA Analysis
5. Ultra-Fast Purification of Small Nucleic Acids
6. Small-Molecule Quantitation Using Automated Single-Shot Averaging MALDI-TOF Mass Spectrometry
7. Maintaining High Sensitivity when Working with Small-Volume Samples
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Got Small RNA

(Date:9/22/2014)... NEW YORK , Sept. 22, 2014 ... US demand for specialty biocides is forecast to ... 2018. In volume terms, demand is projected to increase ... the same period. A strong rebound in construction expenditures ... and paint and coatings markets, while growth in consumer ...
(Date:9/22/2014)... DUBLIN , Sept. 22, 2014 ... the "Global and Chinese Microcrystalline Cellulose Industry Report ... The Global and Chinese Microcrystalline Cellulose Industry Report 2014 ... state of the global microcrystalline cellulose industry with a ... provides a basic overview of the industry including definitions, ...
(Date:9/22/2014)... FierceBiotech today announced its annual “Fierce ... of 2014. FierceBiotech editors John Carroll and Damian ... winners. Those selected are identified as some of ... , "It's always a lot of fun ... companies," said Carroll. "This year in particular you'll find ...
(Date:9/22/2014)... , Sept. 22, 2014 Synageva ... ), a biopharmaceutical company developing therapeutic products for ... Robert Bazemore as Chief Operating Officer.  Reporting ... and Chief Executive Officer, Robert will oversee the ... program, sebelipase alfa for LAL Deficiency, and will ...
Breaking Biology Technology:US Biocides Market 2US Biocides Market 3Global and Chinese Microcrystalline Cellulose Industry Report 2014 2FierceBiotech Announces Annual “Fierce 15” Winners of 2014 2Synageva BioPharma Appoints Robert Bazemore As Chief Operating Officer 2Synageva BioPharma Appoints Robert Bazemore As Chief Operating Officer 3Synageva BioPharma Appoints Robert Bazemore As Chief Operating Officer 4
... Last night, as I watched my brothers set off marginally ... checked e-mail since Sunday morning, and my mobile phone was ... of July weekend, the mid-summer respite, and I felt anything ... hours even over a holiday weekend comes with ...
... as businesses seek to protect and enhance the value ... result of the business' products and services. Each year, ... many of them for federal trademark registration. Indeed, applications ... good leading indicator of economic conditions (a steep drop ...
... Madison, Wis. The National Institutes of ... a $4 million Fast-Track SBIR grant from the ... Diseases. , ,Under this grant, Stratatech will work ... substitute with enhanced antimicrobial and angiogenetic properties for ...
Cached Biology Technology:Declaring independence from technology (sort of) 2Declaring independence from technology (sort of) 3Why file for trademark protection? 2Why file for trademark protection? 3Why file for trademark protection? 4
(Date:9/22/2014)... in recent years has been identified as the plant,s ... aroma also summons beneficial insects to the rescue. , ... the environment via the emission of volatile organic compounds, ... wasps to come to the plant that is being ... Dr. Michael Kolomiets, Texas A&M AgriLife Research plant pathologist ...
(Date:9/22/2014)... in a plant,s biological clock one that modulates ... , Transcription factors, or genetic switches, drive gene expression ... light, rain, soil quality, or even animals grazing on ... one, called FBH1, that reacts to temperature tweaking ... keeping it on a consistent track. , "Temperature helps ...
(Date:9/22/2014)... CITY (September 22, 2014)The Brain & Behavior ... award of NARSAD Young Investigator Grants valued ... of the world,s most promising young scientists. ... groundbreaking neurobiological research will seek to identify ... for psychiatric disorders that affect one in ...
Breaking Biology News(10 mins):Mown grass smell sends SOS for help in resisting insect attacks, researchers say 2Mown grass smell sends SOS for help in resisting insect attacks, researchers say 3Genetic switch regulates a plant's internal clock based on temperature 2Brain & Behavior Research Foundation awards 200 NARSAD Young Investigator grants 2
... for cystic fibrosis, HIV-fighting ,Trojan horses, new pharmaceuticals from ... to discover medications of the future. ,On 24 August, ... an international conference in Gothenburg, Sweden. In ... to investigate, and eventually control, the behavior of cells. ...
... researcher is challenging health standards that consider nitrates and ... research suggests that although there are negative health effects ... nitrates in groundwater, nitrates and nitrites -- as they ... Nitrate and nitrite are naturally occurring ions associated ...
... visceral fat, more commonly called belly fat, is known to ... causes visceral fat to accumulate? The culprit is likely ... hormone balance that occurs during the menopause transition, according to ... factors we analyzed that could possibly account for the increase ...
Cached Biology News:10th Functional Genomics: Chemical Biology 2009 2Research supports calls to study health benefits of nitrate, nitrite 2Increase in visceral fat during menopause linked with testosterone 2
... of 3 siRNAs specifically targeted to your ... product. The siTrio reagent guarantees you get ... gene when used under standard conditions (100 ... optimal transfection with one of B-Bridges controls). ...
... a cocktail of 3 siRNAs specifically targeted ... most popular product. The siTrio reagent guarantees ... your target gene when used under standard ... confirmation of optimal transfection with one of ...
... has improved stability and convenience from the former ... has overcome this problem but also produced some ... the formulation of HRP conjugated antibody from lyophilized ... change, the stability of the conjugate has improved ...
... suitable for a wide range of molecular ... Pulsed Field Gel Electrophoresis (PFGE). Due to ... Multi ABgarose can effectively separate large DNA ... turn means reduced band diffusion, a problem ...
Biology Products: