Navigation Links
Fluorescently Labeling Long dsRNA with the Silencer siRNA Labeling Kit


Although the Silencer siRNA Labeling Kits were developed for labeling siRNA, they can be readily adapted for labeling long dsRNA. The following protocol can be used for this application. See "Visualizing Introduced dsRNA in Cells" for experimintal data using dsRNA labeled in this manner.

Note: Limit the exposure to light for entire procedure.

  1. Add the following reagents in order:

20 l nuclease-free water
5 l 10X Labeling Buffer
20 l dsRNA*
5 l Labeling Dye
50 l Total

  1. Incubate at 37C for 1 hour.
  2. Add 5.0 l (0.1 vol) 5 M NaCl.
  3. Add 125 l (2.5 vol) cold 100% EtOH and mix thoroughly.
  4. Incubate at -20C for 20-30 min.
  5. Centrifuge at top speed at 4C for 20 min.
  6. Remove supernatant and wash pellet with 100 l 70% EtOH.
  7. Centrifuge at top speed for 5 min at room temperature.
  8. Remove supernatant.
  9. Air dry pellet.
  10. Suspend labeled RNA in 20 l (original volume) of nuclease-free water .


*We have successfully labeled 40 to 80 pmol of dsRNA, 400 to 1200 nt in length, with this protocol. The amount of dsRNA per reaction may need to be optimized according to the size and concentration of the dsRNA. In general, we obtain a maximum silencing effect with a final concentration of 10 nM labeled dsRNA.



Visualizing Introduced dsRNA in Cells

One way to further understand the mechanism of RNAi is to track the introduced dsRNA in live cells. Fluorescent labeling is commonly used by scientists to localize macromolecules, but it was not known whether the incorporation of a fluorophore into dsRNA would affect its ability to induce gene silencing. To address this question, we used the Silencer siRNA Labeling Kit to couple the fluorescent label, Cy3, to long dsRNA prepared with the MEGAscript RNAi Kit and then compared labeled dsRNA activity to that of unlabeled.

Figure 1A shows that the same powerful silencing effect was obtained with both labeled and unlabeled dsRNA specific for hrp48 or U2AF50 gene products. No cytotoxic effect was associated with the fluorescent moiety and the expression level of a non-related control protein, Pab 1, was not affected (Figure 1A). The specific silencing of hrp48 and U2AF50 proteins was also confirmed by immunofluorescence microscopy (data not shown). Direct observation of the Cy3 fluorescence demonstrated that both hrp48 and U2AF50 labeled dsRNA were taken up equivalently into L2 cells and localized to discrete perinuclear foci in the cytoplasm (Figure 1B). This localization is very similar to what was previously reported for siRNA in mammalian cells (Byron et al., 2002). Finally time course studies showed that the RNAi effect was maintained in cultured Drosophila cells for up to 10 days and that labeled dsRNA was passed on to daughter cells during cell division (data not shown).

Figure 1. Fluorescently Labeled dsRNA Retains Functionality. The MEGAscript RNAi Kit was used to generate full-length dsRNA to the Drosophila hrp48 and U2AF50 genes from the corresponding cDNA constructs. A portion of the dsRNA was labeled with Cy3 using the Silencer siRNA Labeling Kit according to the above protocol. 1x106 Schneider's Drosophila L2 cells were grown in 6 well plates in serum free medium and directly treated with 10 nM of dsRNA (no transfection is required in these cells). Three days after treatment, (A) hrp48 and U2AF50 protein levels were assessed by Western blot and (B) cells were analyzed by fluorescence microscopy. BLUE: DAPI stained nuclei. RED: Cy3 labeled U2AF50 dsRNA.


Related Articles
RNAi in Non-Mammalian Cultured Cells


back to top


Ordering Information
Cat# Product Name Size 1626 MEGAscript RNAi Kit 20 rxns 1632 Silencer siRNA Labeling Kit - Cy3 65 g labeled siRNA 1634 Silencer siRNA Labeling Kit - FAM 65 g labeled siRNA
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. New Microarray Labeling Kit for Preparing cDNA Probes
2. aRNA Synthesis and Labeling for Array Analysis
3. Amino Allyl Labeling for Array Analysis
4. Efficient Europium Labeling of Lymphokine-Activated Killer Cells Using the Gene Pulser II with RF Module, Rev C
5. USB OptiKinase Eliminates Base Bias in Labeling of Oligonucleotide 5-Ends
6. Silencer Validated siRNAs
7. pSilencer 4.1-CMV: Versatile Vectors for Expression of siRNA, miRNA, and mRNA
8. Selecting siRNA Sequences to Incorporate into the pSilencer Vectors
9. Your Data: Use of pSilencer 1.0-U6 siRNA Expression Vector in the Analysis of Synaptotagmin Location and Function
10. YOUR DATA: Direct Real-time RT-PCR on Sympathetic Neuron Lysates Silencing of Focal Adhesion Kinases with Silencer Validated siRNAs
11. YOUR DATA: Direct Real-time RT-PCR on Sympathetic Neuron Lysates Silencing of Focal Adhesion Kinases with Silencer Validated siRNAs
Post Your Comments:
(Date:1/15/2014)... , Jan. 15, 2014 ­ RedBrick Health , ... change technology, today announces that EmblemHealth , the ... wellness company, is now providing the RedBrick Compass health ... program, to all of its members. EmblemHealth is among ...
(Date:1/15/2014)... 2014 Two champions of science, technology, ... of an annual competition for middle and high school ... innovative STEM study. The competition presents students with real-world ... Engineering Aptitude, Mathematics, and Sciences is a program ...
(Date:1/15/2014)... (PRWEB) January 15, 2014 Freeslate, ... solutions, today announced that Lupin Limited, one of India’s ... CM Protégé PharmD System for high throughput ... India, is focused on a wide range of quality, ...
(Date:1/14/2014)... 2014 Carahsoft and CDS Federal Services ... 2014 at 2pm EST (11am PST), “Natural Language Processing: ... on how technology can turn raw, heterogeneous data into ... agencies. The online webinar will last approximately one hour. ...
Breaking Biology Technology:EmblemHealth Selects RedBrick Health to Power its Next-Generation Health Engagement and Behavior Change System 2EmblemHealth Selects RedBrick Health to Power its Next-Generation Health Engagement and Behavior Change System 3Technology Student Association Partners with Leaders in STEM Education Advocacy for Annual Competition 2Technology Student Association Partners with Leaders in STEM Education Advocacy for Annual Competition 3Technology Student Association Partners with Leaders in STEM Education Advocacy for Annual Competition 4Technology Student Association Partners with Leaders in STEM Education Advocacy for Annual Competition 5Lupin Selects Freeslate’s CM Protégé PharmD System to Accelerate Polymorph Screening for Drug Development 2Lupin Selects Freeslate’s CM Protégé PharmD System to Accelerate Polymorph Screening for Drug Development 3Webcast - Natural Language Processing: Converting Raw Data into Actionable Knowledge – Hosted by Carahsoft and CDS Federal Services 2
... do Dr. Peper, Dr. Sigmund Freud and Arnold Schwarzenegger ... be surprised to learn that Europeans love Dr. Peper. ... Erik Peper, President of the Biofeedback Foundation of Europe ... Dr. Erik Peper opens the 13th Annual Conference of ...
... Presents on Therapy Development StrategyCAMBRIDGE, Mass., Feb. 17 ... has announced the 2009 launch of its popular ... seminar is designed to inform and update patients, ... to discover and develop effective treatments for amyotrophic ...
... Inc. (OTC Bulletin Board: GNPH) ("Genesis" or the,"Company"), a ... Republic of China, today announced its financial results for ... 31, 2008. , , Second Quarter ... Revenue was $32.9 million, up 24.1% from the corresponding ...
Cached Biology Technology:Dr. Peper, Dr. Freud and Arnold Schwarzenegger 2Seminar Will Bring ALS Researchers to Patient Groups 2Seminar Will Bring ALS Researchers to Patient Groups 3Genesis Pharmaceuticals Reports Results for the Second Quarter of its Fiscal Year 2009 2Genesis Pharmaceuticals Reports Results for the Second Quarter of its Fiscal Year 2009 3Genesis Pharmaceuticals Reports Results for the Second Quarter of its Fiscal Year 2009 4Genesis Pharmaceuticals Reports Results for the Second Quarter of its Fiscal Year 2009 5Genesis Pharmaceuticals Reports Results for the Second Quarter of its Fiscal Year 2009 6Genesis Pharmaceuticals Reports Results for the Second Quarter of its Fiscal Year 2009 7Genesis Pharmaceuticals Reports Results for the Second Quarter of its Fiscal Year 2009 8Genesis Pharmaceuticals Reports Results for the Second Quarter of its Fiscal Year 2009 9Genesis Pharmaceuticals Reports Results for the Second Quarter of its Fiscal Year 2009 10Genesis Pharmaceuticals Reports Results for the Second Quarter of its Fiscal Year 2009 11Genesis Pharmaceuticals Reports Results for the Second Quarter of its Fiscal Year 2009 12Genesis Pharmaceuticals Reports Results for the Second Quarter of its Fiscal Year 2009 13Genesis Pharmaceuticals Reports Results for the Second Quarter of its Fiscal Year 2009 14Genesis Pharmaceuticals Reports Results for the Second Quarter of its Fiscal Year 2009 15Genesis Pharmaceuticals Reports Results for the Second Quarter of its Fiscal Year 2009 16
(Date:4/17/2014)... April 17, 2014The development of stem cell therapies to ... to characterize stem cell populations based on cell surface ... a new marker that is highly expressed in a ... blood, which they describe in an article in ... Ann Liebert, Inc., publishers. The article is available free ...
(Date:4/17/2014)... Marc Andr Gauthier and Professor Luca Razzari of ... been awarded large grants from the John R. ... Innovation (CFI) for the acquisition of state-of-the-art biotech ... added matching grants from the Ministre de l,Enseignement ... de la Technologie (MESRST). These new laboratories will ...
(Date:4/17/2014)... news release is available in German . ... us to recognise objects; they also provide us with a ... run, turn around, fall or sit still in a car ... motion trace on our retinas. Seemingly without effort, our brain ... maintain a stable position and a steady gaze during our ...
Breaking Biology News(10 mins):New state-of-the-art biotech and nanotech equipment for INRS 2How vision makes sure that little fish do not get carried away 2How vision makes sure that little fish do not get carried away 3How vision makes sure that little fish do not get carried away 4
... NARRAGANSETT, RI -- June 7, 2011 -- Sunshine Menezes, executive ... and 2010 Grantham Prize winner Alanna Mitchell announced the 2011 ... the Environment. James Astill of The Economist will receive US$75,000 ... a commanding 8-part special report on the state of global ...
... in billionths of a meter, self-assembling nano-sized devices designed ... are revolutionizing medicine by improving drug solubility and bio-distribution, ... and enabling membrane barriers to be crossed as well ... Self-assembling nano devices are now enlisted in the ...
... turns out, humans aren,t the only organisms that turn to ... four different bacteria that actually can live on caffeine. ... was found in a flowerbed outside a UI research laboratory. ... understanding of how the process works -- could in the ...
Cached Biology News:$75,000 Grantham Prize winner announced 2$75,000 Grantham Prize winner announced 3Super-mini vehicles carry therapeutics and imaging agents into body with mega results 2University of Iowa researchers identify caffeine-consuming bacterium 2
Sf21 Insect Cells (Frozen) in Max-XP Medium...
Lyophilized solid. Suitable for evaluating functional activity of human complement components and for other research requiring a high level of hemolytic activity. ...
... arrays have been developed for affinity ... A variety of ProteinChip chemical surfaces ... to their unique biochemical properties. Each ... side-by-side sample comparison, and the chips ...
... been developed for affinity capture of ... of ProteinChip chemical surfaces allow differential ... unique biochemical properties. Each chip has ... comparison, and the chips are color-coded ...
Biology Products: