Protein activity and gene expression within a cell hold major significance for researchers. Of particular interest are the changes in cellular mRNA levels, this is because these changes directly correlate to their corresponding protein levels.
However, there are exceptions to the rule. Gene expression is investigated by many scientists by observing the response of mRNA molecules to genetic manipulations. This is usually achieved by using several different techniques such as RT-PCR, Northern blotting, and nuclease protection assays.
Advantages of Ribonuclease Protection Assays
Ribonuclease protection assays (RPA) have several benefits compared to other techniques, such as Northern blot, which rely on binding RNA to a solid support. This is because, even when there is low abundance of mRNA in a total RNA solution, it can still be readily detected by RPA assays.
Using a nonradioactive chemiluminescent approach, RPA assays provide more sensitivity even when small amounts of total RNA are used. Syngene’s G:BOX iChemi XR & XT automated chemiluminescent image analyzers are well-suited for non-radioactive RPA work.
While the cooled CCD cameras in these analyzers enable long integration times with low background noise, the analyzers themselves provide hands-free, automated series image capture.
Materials and methods
Generating and hybridizing non-radioactively labeled RNA
In vitro transcript RNA probes are synthesized and labeled with biotin, a non-radioactive marker simultaneously, as shown in Figure 1.
Figure 1. Schematic of RPA protocol. Probes (in 3-10 fold molar excess to the total RNA) and total RNA are hybridized (Step 1). The hybridized probes are then treated with RNase A and T1 to degrade any single-stranded RNA. Labeled probe and complementary RNA complex is protected from RNase digestion and is run on denaturing polyacrylamide gel (Steps 2 and 3). The polyacrylamide gel is transferred to a nylon membrane. The membrane is treated with chemiluminescence before being visualized (Steps 4, 5 and 6). Image adapted from Piercenet.com
A detection system such as the BrightStarTM BioDetectTM Kit (Ambion, Huntingdon, UK) is employed to visualize hybridization of the biotinylated RNA probe. After placing the blot in the G:BOX iChemi XR/XT’s light tight darkroom, images are captured with the GeneSnap image acquisition software.
Then, G:BOX iChemi’s Extended Dynamic Range (EDR) is applied by combining up to 65,000 captured light exposures. The EDR is used to detect the faint chemiluminescent bands. Using the GeneTools software, the optimized image can be automatically analyzed.
Researchers have reported that RPA blot images having a high signal-to-noise ratio, can be produced by using a G:BOX iChemi XR/XT with a chemiluminescent RPA. This has helps the researchers in the identification of mRNA specific to their target proteins from as little as 5 - 50 ng mRNA, in a solution of 5-20 μg total RNA.
Syngene’s G:BOX iChemi XR/XT offers a sensitive technique for effective detection of small volumes of mRNA in a non-radioactive RPA. The software enables series image capture, allowing EDR researchers to save hands-on time while still producing a perfectly exposed RPA blot image.
Quantitative data can be generated by analyzing this image with the GeneTools software, a task that is not easy to perform when using X-ray film.
Syngene are a division of the Synoptics Group, founded in 1985 by imaging experts from the University of Cambridge. At Syngene we live and breathe image analysis because products specifically for gel documentation and fluorescence/chemiluminescence imaging are all we’ve ever focused on developing.
Our other divisions in the Synoptics Group, Synbiosis and Syncroscopy, develop imaging solutions for microbial and microscopy applications so we are complete life science imaging specialists. Synoptics Health focuses on imaging techniques within the clinical environment.
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