Navigation Links
Cells-to-cDNA II Applications

Quantitation of siRNA Target Gene Expression


RT-PCR is one of the easiest ways to confirm gene profiling and gene function analyses generated by methods such as array analysis and siRNA experiments, especially when there are many samples to analyze. However, there is one catch. For RT-PCR experiments, total RNA must be isolated first. Why not skip this step? Ambion scientists now demonstrate the use of the Cells-to-cDNA II Kit to produce real time RT-PCR data quickly, with minimal effort. Cells-to-cDNA cell lysates can be used for RT-PCR directly without RNA isolation. In this article, we show how the Cells-to-cDNA II Kit can be used to assess siRNA experiments for the development of an optimized RNA interference protocol. In addition to analyzing siRNA data, Cells-to-cDNA can be used to process FACS and LCM samples. The kit has also been adapted to 96 well plate automated processing. See "Automated Cells-to-cDNA Protocol" for the procedure.


Experimental Set-up: siRNA Transfection
HeLa cells were transfected with siRNA and an automated protocol for the Cells-to-cDNA II Kit was used to look at specificity of the siRNA effect and optimal cell concentration.

HeLa cells were examined under three experimental conditions: 1) cells that were transfected with a GAPDH siRNA, 2) cells that were transfected with a negative control siRNA and 3) untransfected cells. To optimize cell plating density fo r transfection of HeLa cells, a 96 well plate format was utilized. The plate was divided into 3 sections. Each section corresponded to one of the three experimental conditions outlined above. Each individual row contained the same number of initial cells. The cells were diluted 2 fold from the top of the plate to the bottom starting with 64,000 cells and ending with 1000 cells. This gave 4 replicates for each experimental condition.

siRNAs were made using the Silencer siRNA Construction Kit (Ambion). Both GAPDH siRNA and negative control siRNA (included in the Silencer siRNA Construction Kit) were transfected into cells at a final concentration of 10 nM using siPORTLipid Transfection Agent (Ambion). Fresh medium was added 4 hours after transfection.


Data Analysis: Using Automated Cells-to-cDNA II and One-Step Real-time RT-PCR
The cell culture plate was transferred to the deck of a Perkin Elmer MultiPROBE II robotic platform 48 hours post transfection. The automated Cells-to-cDNA II protocol was performed followed by one-step real-time RT-PCR using a TaqMan probe and primers for GAPDH. The robot removed the growth media, washed the cells with PBS, and then added 100 l of Cell Lysis Buffer. The plate was moved to a heating tile, which brought the samples to 75C for 12 min, simultaneously disrupting the cells and inactivating RNases. The samples were treated with DNase I followed by heat inactivation. Lysate (5 l) was transferred robotically to a 96 well PCR plate containing 20 l of a one-step real-time RT-PCR master mix.

This analysis determined that the optimal cell concentration necessary to induce maximum interference was 2000 cells per well (Figure 1). Data clearly show that the optimal number of HeLa cells plated in this experiment was 2,000 cells (Figure 1). At this plating density, cells transfected with the siRNA targeting GAPDH expressed only 30% the GAPDH levels of those transfected with the negative control siRNA.

Figure 1. Optimization of siRNA Transfection Using Cells-to-cDNA II Automated Protocol. The Y axis shows GAPDH expression after transfection with GAPDH siRNA as a percent of expression after transfection with the negative control siRNA. A standard curve for GAPDH expression was used to normalize the data. An ABI 7900 was used to perform realtime PCR.


Ambion's Silencer siRNA products are described in more detail in, "Cost Effective High Potency siRNAs". For more information about the Cells-to-cDNA II automated protocol, see "Automation of One-Step RT-PCR Using Ambion's Cells-to-cDNA II Kit and the MultiPROBE II HT Liquid Handling System" or contact our technical service department by email at http://www.ambion.com/contact/techserv_email.html or phone at (800) 888-8804 option 2. Our customers frequently develop new and interesting adaptations of our Cells-to-cDNA technology. Do you have an unusual Cells-to-cDNA story? If so we would like to know!

TaqMa n is a registered trademark of Applied Biosystems.


back to top


Ordering Information
Cat# Product Name Size 1620 Silencer siRNA Construction Kit 15 siRNA synthesis rxns 1722 Cells-to-cDNA II Kit 40 rxns 1723 Cells-to-cDNA II Kit 100 rxns 4502 siPORT Amine Transfection Agent 0.4 ml 4503 siPORT Amine Transfection Agent 1 ml
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Cells-to-cDNA II Using FACS Sorted Cells
2. Your Data: Detection of Oocyte-specific Gene Expression Using Ambions Cells-to-cDNA Kit
3. A Versatile Power Supply for All Electrophoresis Applications
4. Highest Possible Transformation Efficiencies for High-Throughput Applications
5. Unique DNA Polymerase Formulation Excels in a Broad Range of PCR Applications
6. High-Throughput System Generates Ultra-Pure PCR Products Suitable for All Applications
7. Optimizing Büchi® Rotary Evaporator Applications
8. Optimizing Büchi® Rotary Evaporator Applications
9. Optimizing Bchi Rotary Evaporator Applications
10. Uses and Applications of X-tremeGENE Q2 Transfection Reagent
11. Uses and Applications of FuGENE 6 Transfection Reagent
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:1/11/2017)... ... January 11, 2017 , ... Photonics industry and ... , are commending the U.S. Congress and President Obama for their recognition of ... of the American Innovation and Competitiveness Act (AICA). , The language of the ...
(Date:1/11/2017)... Yorba Linda, Ca (PRWEB) , ... January 11, ... ... antibiotic resistance in pathogens are observed in clinical settings, it is becoming increasingly ... ecological problem. An over-reliance on culture-based methods, the standard in the study of ...
(Date:1/11/2017)... ... January 11, 2017 , ... For many fledging startups, good ... challenges young businesses face. With the second installment of Quorum’s newest signature program, ... DiMasi, Founder and Principal of interactive design agency, P’unk Ave. , For ...
(Date:1/11/2017)... , ... January 11, 2017 , ... ... development professional has joined its team. Bernhard Bartylla will lead European initiatives for ... with Bernhard to introduce ACOMP and ARGEN to European manufacturers and researchers. Bernhard ...
Breaking Biology Technology:
(Date:12/15/2016)... AUBURN HILLS, Mich. , Dec. 15, 2016 /PRNewswire/ ... simply unlocking car doors or starting the engine. Continental ... 2017 in Las Vegas . Through ... PASE (Passive Start and Entry) and biometric elements, the ... the field of vehicle personalization and authentication. ...
(Date:12/8/2016)... Research Future published a half cooked research report on Mobile Biometric ... Market is expected to grow over the CAGR of ~35% during ... ... Mobile Biometric Security and Service Market is increasing at a ... security from unwanted cyber threats. The increasing use of mobile device ...
(Date:12/7/2016)... TEL AVIV, Israel , December 7, 2016 ... year with the expansion of its patent portfolio, which grew to over ... , , ... led by its recently filed patent entitled " System, Device, ... which covers technology that enables device makers to forego costly hardware components ...
Breaking Biology News(10 mins):