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:7/30/2015)... 2015   Senomyx , Inc. (NASDAQ: SNMX ... to discover, develop, and commercialize novel flavor ingredients for ... financial results for the second quarter 2015. ... remain on track to achieve our commercial and financial ... of the Company. "Since our last quarterly earnings report, ...
(Date:7/30/2015)... , July 30, 2015  Discovery Laboratories, Inc. ... has received the second $1.0M tranche under a ... (SBIR) grant valued at up to $3.0 million ... KL4 surfactant as a potential medical countermeasure to ... Labs was awarded an initial $1.0 million under ...
(Date:7/29/2015)... , July 30, 2015 ... la santé, publie ses résultats pour le premier ... commente les résultats. Visionner l,interview ... http://www.eurobusinessmedia.com/ceo-direct/sanofi/sanofi-h1-2015-results-interview-with-ceo-olivier-brandicourt?utm_source=ceo-direct&utm_medium=wire Au sommaire de ... - Moteurs de croissance - ...
(Date:7/29/2015)... July 29, 2015 /PRNewswire/ - BioAmber Inc. (NYSE: BIOA ... Company,s Chief Financial Officer has resigned.  The Company has ... Officer who retired in December 2014, as full time ... permanent successor.     Andy Ashworth began ... ensure a smooth transition.  Andy was the Company,s CFO ...
Breaking Biology Technology:SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 2SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 3SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 4SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 5SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 6SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 7SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 8SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 9Discovery Labs Receives $1.0 million under an SBIR Grant in Continuing Support of the Development of Aerosolized KL4 Surfactant to Address Radiation-Induced Lung Injury 2Discovery Labs Receives $1.0 million under an SBIR Grant in Continuing Support of the Development of Aerosolized KL4 Surfactant to Address Radiation-Induced Lung Injury 3BioAmber Announces Change in Chief Financial Officer 2BioAmber Announces Change in Chief Financial Officer 3
... Reportlinker.com announces that a new ... its catalogue: DNA ... Purchase Plans and The Impact of ... http://www.reportlinker.com/p0681348/DNA-Microarray-Market-Trends-Installed-Base-Applications-Purchase-Plans-and-The-Impact-of-Next-Gen-Sequencing.html#utm_source=prnewswire&utm_medium=pr&utm_campaign=In_Vitro_Diagnostic DNA ...
... Novartis announced today new pivotal Phase III data showing ... (SJIA) were able to substantially reduce their use of ... of commencing treatment with ACZ885 (canakinumab) (p<0.0001)[1]. ... primary endpoints, will be presented on November 9th at ...
... Jennerex, Inc., a private clinical-stage biotherapeutics company focused ... oncolytic products for cancer, today presented final data from ... in patients with advanced liver cancer showing a statistically ... dose group versus the low dose group.  The final ...
Cached Biology Technology:DNA Microarray Market Trends (Installed Base, Applications, Purchase Plans and the Impact of Next-Gen Sequencing) 2DNA Microarray Market Trends (Installed Base, Applications, Purchase Plans and the Impact of Next-Gen Sequencing) 3DNA Microarray Market Trends (Installed Base, Applications, Purchase Plans and the Impact of Next-Gen Sequencing) 4DNA Microarray Market Trends (Installed Base, Applications, Purchase Plans and the Impact of Next-Gen Sequencing) 5DNA Microarray Market Trends (Installed Base, Applications, Purchase Plans and the Impact of Next-Gen Sequencing) 6DNA Microarray Market Trends (Installed Base, Applications, Purchase Plans and the Impact of Next-Gen Sequencing) 7DNA Microarray Market Trends (Installed Base, Applications, Purchase Plans and the Impact of Next-Gen Sequencing) 8DNA Microarray Market Trends (Installed Base, Applications, Purchase Plans and the Impact of Next-Gen Sequencing) 9Novartis Phase III Study Shows ACZ885 Helped Substantially Reduce Steroid Use in 45% of Patients With Serious Form of Childhood Arthritis 2Novartis Phase III Study Shows ACZ885 Helped Substantially Reduce Steroid Use in 45% of Patients With Serious Form of Childhood Arthritis 3Novartis Phase III Study Shows ACZ885 Helped Substantially Reduce Steroid Use in 45% of Patients With Serious Form of Childhood Arthritis 4Novartis Phase III Study Shows ACZ885 Helped Substantially Reduce Steroid Use in 45% of Patients With Serious Form of Childhood Arthritis 5Novartis Phase III Study Shows ACZ885 Helped Substantially Reduce Steroid Use in 45% of Patients With Serious Form of Childhood Arthritis 6Novartis Phase III Study Shows ACZ885 Helped Substantially Reduce Steroid Use in 45% of Patients With Serious Form of Childhood Arthritis 7Jennerex Presents Final Data From JX-594 Randomized Phase 2 Clinical Trial Showing Statistically Significant Survival Benefit in Patients With Advanced Liver Cancer 2Jennerex Presents Final Data From JX-594 Randomized Phase 2 Clinical Trial Showing Statistically Significant Survival Benefit in Patients With Advanced Liver Cancer 3Jennerex Presents Final Data From JX-594 Randomized Phase 2 Clinical Trial Showing Statistically Significant Survival Benefit in Patients With Advanced Liver Cancer 4Jennerex Presents Final Data From JX-594 Randomized Phase 2 Clinical Trial Showing Statistically Significant Survival Benefit in Patients With Advanced Liver Cancer 5Jennerex Presents Final Data From JX-594 Randomized Phase 2 Clinical Trial Showing Statistically Significant Survival Benefit in Patients With Advanced Liver Cancer 6
(Date:7/31/2015)... , 31 de julho de 2015 A ... www.icg-10.org ) será realizada pela BGI de 22-25 de outubro ... A conferência está celebrando seu 10 o. ... a ICG se tornou uma das reuniões mais influentes ... encontros científicos mais dinâmicos, entusiastas e prazerosos. ...
(Date:7/31/2015)... NXT-ID, Inc. (NASDAQ: NXTD ) ("NXT-ID" or ... growing mobile commerce market and creator of the Wocket® ... 62/198989 for ELECTRONIC CRYPTO-CURRENCY MANAGEMENT METHOD AND ... advance crypto-currencies such as Bitcoin into the consumer market ... to manage all payments.  The technology ...
(Date:7/27/2015)... , July 27, 2015   Zynx Health ... experience-based clinical improvement solutions, today announced that its ... now available on Android smartphones and tablets. With ... post-care organizations can use ZynxCarebook to securely exchange ... condition, streamline care transitions to other care settings, ...
Breaking Biology News(10 mins):A 10a. Conferência Internacional sobre Genômica (ICG-10) será em outubro 2A 10a. Conferência Internacional sobre Genômica (ICG-10) será em outubro 3NXT-ID Patents Electronic Crypto-Currency Management Technology 2NXT-ID Patents Electronic Crypto-Currency Management Technology 3NXT-ID Patents Electronic Crypto-Currency Management Technology 4Zynx Health Adds Android Device Support to ZynxCarebook Solution 2Zynx Health Adds Android Device Support to ZynxCarebook Solution 3
... VIB and Ghent University, both in Flanders, Belgium, is ... worth 750 000 euro. The Krber Foundation annually awards ... Friml gets the prize for his research into the ... the genetic, molecular and cellular processes that direct the ...
... have discovered components of the bovine mastitis-causing bacterium, Streptococcus ... This discovery could lead the way to finally developing a ... nearly 200M per year, requires the large scale use of ... A solution to this problem will be an important contribution ...
... the world,s largest living reptile - the estuarine crocodile - ... by huge stretches of ocean despite being a poor swimmer ... ecologists. Publishing their new study in the British Ecological Society,s ... a surfer catching a wave, the crocodiles ride ocean currents ...
Cached Biology News:VIB-UGent scientist Jiri Friml receives Koerber European Science Award 2010 2New discovery could aid development of elusive bovine mastitis vaccine 2Crocodiles ride ocean currents for ocean travel 2Crocodiles ride ocean currents for ocean travel 3
... silencing experiments often call for critical, ... Transfection Agent refines siRNA transfection protocols ... lipid-based formulation can be used to ... are subcultured without increased cytotoxicity. ...
... The CHEMICON® CaspaTag™ Pan-Caspase In Situ ... detection of active caspases in cells undergoing ... only. Not for use in diagnostic or ... Assay Kits use a novel approach to ...
... INTERFERin is a new-generation siRNA transfection ... efficiency at 1 nM siRNA in a ... with excellent cell viability. Using low siRNA ... effects observed when transfecting siRNA at higher ...
... Cell and Tissue Staining Kits are intended ... range of histological and cytological specimens. These ... antibodies manufactured by either R&D Systems or ... on the formation of the Avidin-Biotin Complex ...
Biology Products: