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:12/24/2014)... 2014 Sigma-Aldrich Corporation (NASDAQ: ... under the Hart-Scott-Rodino Antitrust Improvements Act (HSR Act) ... 22, 2014, thereby completing the U.S. HSR Act ... of the Company by Merck KGaA, Darmstadt, Germany.  ... condition to closing the transaction, which remains subject ...
(Date:12/22/2014)... , Dec. 22, 2014 /PRNewswire/ - RepliCel Life ... regenerative medicine company focused on the development of autologous ... out of the University of Calgary in conjunction with ... Carolina, which further validates the company,s ongoing clinical research ... effects of pattern baldness. The paper entitled ...
(Date:12/22/2014)... ITRA Global, one of the largest organizations ... estate, has further expanded its global reach with the ... Western Australia, reports Mylinda Vick, CCIM, Chairman of the ... / ACORPP (Australian Corporate Property and Projects) , is ... services to a wide range of clients in the ...
(Date:12/19/2014)... Dec. 19, 2014 Naurex Inc., a biopharmaceutical ... for diseases of the central nervous system, today announced ... executive officer, will present at the 33 rd ... take place at 3:00 p.m. PST on Tuesday, January ... San Francisco, Calif. ...
Breaking Biology Technology:Acquisition of Sigma-Aldrich by Merck KGaA, Darmstadt, Germany, Receives U.S. Antitrust Clearance 2Acquisition of Sigma-Aldrich by Merck KGaA, Darmstadt, Germany, Receives U.S. Antitrust Clearance 3Newly Released Peer-Reviewed Publication Further Validates RepliCel's Use of Dermal Sheath Cup Cells to Treat Pattern Baldness 2Newly Released Peer-Reviewed Publication Further Validates RepliCel's Use of Dermal Sheath Cup Cells to Treat Pattern Baldness 3Newly Released Peer-Reviewed Publication Further Validates RepliCel's Use of Dermal Sheath Cup Cells to Treat Pattern Baldness 4Newly Released Peer-Reviewed Publication Further Validates RepliCel's Use of Dermal Sheath Cup Cells to Treat Pattern Baldness 5ITRA Global Continues International Expansion in Perth and Brisbane, Australia 2Naurex to Present at 33rd Annual J.P. Morgan Healthcare Conference 2
... , D.J. Lowrie Jr. ,Department of Cell Biology, ... Medicine, Cincinnati, OH 45267 , , Transfection of ... of,liposome-based reagents. Here we compare the transfection efficiency ... both LipoTAXI ,transfection reagent ...
... Taq DNA polymerase , makes hot start simple and efficient , ... , , , Jonathan Eads ... Stratagene , , , ... Taq , DNA polymerase , ...
... and effort needed for,transformationswithout compromise! , Anna Waesche ... Alan Greener ,Stratagene , Stratagene introduces competent cells from ... easy-to-use, single-reaction format that,yield the supercompetent efficiency of 1 ... the competent efficiency of 1 x 10 8 ...
Cached Biology Technology:Transfection of Green Fluorescent Protein into Human Adrenalcarcinoma Cells 2Transfection of Green Fluorescent Protein into Human Adrenalcarcinoma Cells 3Transfection of Green Fluorescent Protein into Human Adrenalcarcinoma Cells 4Greater Amplification Specificity with New Hot Start PCR Enzyme 2Greater Amplification Specificity with New Hot Start PCR Enzyme 3Greater Amplification Specificity with New Hot Start PCR Enzyme 4Greater Amplification Specificity with New Hot Start PCR Enzyme 5Greater Amplification Specificity with New Hot Start PCR Enzyme 6Greater Amplification Specificity with New Hot Start PCR Enzyme 7XL10-Gold Cells Supplied in New Single-Reaction Format 2XL10-Gold Cells Supplied in New Single-Reaction Format 3XL10-Gold Cells Supplied in New Single-Reaction Format 4
(Date:12/11/2014)... 09, 2014 Research and Markets ... ( http://www.researchandmarkets.com/research/9ql3kr/biometrics_market ) has announced the addition ... report to their offering. ... the adoption of multimodal biometric systems. Multimodal biometric ... individual for verification and identification purposes. This helps ...
(Date:12/10/2014)... , Dec. 8, 2014 You,ve been here ... account but can,t remember your password, site key or the ... was your first grade teacher? Today, ... the app that will finally put an end to ... 1U TM . 1U leverages a user,s smartphone ...
(Date:12/10/2014)... 2014 CIE San Diego has launched an ... connective tissue that enhances care coordination among social service ... client-level information; earned a second $1 million grant from ... seniors aging in community and; will be recognized as ... th 4-6p. CIE San Diego today ...
Breaking Biology News(10 mins):Biometrics Market in India 2015-2019: Key Vendors are 3M Cogent, NEC, Safran and Suprema 2The Password is Finally Dead: Launch of 1U Mobile App Eliminates Need for All Usernames and Passwords 2The Password is Finally Dead: Launch of 1U Mobile App Eliminates Need for All Usernames and Passwords 3Community Information Exchange San Diego Launches Pilot, Secures $1 Million Expansion Grant, and Becomes a "Live Well San Diego" Partner 2Community Information Exchange San Diego Launches Pilot, Secures $1 Million Expansion Grant, and Becomes a "Live Well San Diego" Partner 3Community Information Exchange San Diego Launches Pilot, Secures $1 Million Expansion Grant, and Becomes a "Live Well San Diego" Partner 4
... (Pink Sheets: ESMT); with its parent company IVI Smart ... that it has,delivered the Company,s newest Super Smart Card(TM), ... Both companies celebrated the delivery of the "I AM"(TM) ... Center in Seoul, Korea,presided over by Mr. Teawon Park, ...
... Center at Washington University School of Medicine increased its ... Genome Sequencer FLX Systems from Roche Diagnostics. With ... Genome Sequencing Center will be able to support a ... ranging from the sequencing of new complex genomes to ...
... that harvests far-red light by making a rare form ... according to a team of researchers who recently sequenced ... State University and Washington University, St. Louis, report in ... of the National Academy of Sciences, that they have ...
Cached Biology News:e-Smart(R) Technologies Delivers Its New Super Smart Card(TM) in South Korea. 2Washington University increases fleet of Roche Genome Sequencer FLX Systems 2Washington University increases fleet of Roche Genome Sequencer FLX Systems 3Researchers decode genetics of rare photosynthetic bacterium 2Researchers decode genetics of rare photosynthetic bacterium 3
Anti-Src-Like Adaptor Protein-2 AVOID FREEZE/THAW CYCLES. Recognizes the ~25 and ~28 kDa forms SLAP-2....
... media for spontaneous ligand immobilization can be coupled ... antigen, or receptor to create an affinity matrix ... couple ligands via primary amines. Affi-Gel 10 gel ... 6.5 to 11; Affi-Gel 15 gel couples acidic ...
pH-dependent fluorescence; Maximum intensity observed in basic solutions....
Anti-Chicken IgY, (IgG) F(ab')2 Fragment Specific....
Biology Products: