A: Yes. Bio Rad precast 15% Mini-Protein TBE-Urea gels (Cat. No. 4566053 and 4566055) are fully compatible with the EZ-QC™ mRNA Cap 1 Efficiency Assay Kit. Please find instructions on running the Bio-Rad gel in the Product manual.
A: As little as 12 pmol of input mRNA can be used for this gel-based assay. Mass spectrometry may require inputs of ≥50 pmol of input mRNA. In addition, samples for mass spec cannot exceed 10-30 nucleotides long, whereas the EZ-QC™ mRNA Cap 1 Efficiency Assay was designed for use on full length mRNAs. mRNAs from 330-8,500 nucleotides in length have been successfully assayed with this method. In addition, LC-MS is expensive to run and operate, and the user is required to provide specific controls for running their samples on the system for system suitability. Mass spec may have difficulty discerning Cap 0 from Cap 1, especially if there are contaminants present in the samples.
A: We recommend using 12 pmol for ease of testing and visualization.
A: Yes, but it is easier and faster to use the magnetic beads. For mRNA smaller than 750 nt, it is advisable to use inputs >24 pmol since precipitation may have reduced recovery.
A: Yes, and it is advisable to add reagents in the order prescribed in the kit manual.
A: 70% ethanol does not need to be made fresh prior to clean-up, but if the percentage of ethanol drifts too low, sample loss may occur.
A: Once samples have been added to the oxidation reaction mixes, all reactions taking place at the bench should be performed at room temperature.
A: The assay requires about 70 minutes of hands-on time, and with incubations and running on a gel, it takes 1 business day to complete from start to finish. After Cap 1 Stop/Loading Dye is added, this provides a stopping point during which samples can be frozen and run on a gel at a later time. After running samples on a gel, the residual volume can be retained for any gel reruns that may be needed. Completed samples can be stored for up to a week total at -20°C.
A: For the 80/20 Cap 1 Control Mix, we do not recommend decreasing the suggested volume. For experimental samples, we recommend running at least 15 µl of the 34 µl final sample volume on a gel. However, as little as 7.5 µl may be run, but depending on the total Cap 0/Cap 1 content of the sample the accuracy of the assay may be impacted.
Depending on gel well capacity, more of the sample can be loaded, but more background bands may be visible as more sample is loaded.
A: Yes, the samples should still be fine to run on a gel, but for consistent and best results store at -20°C.
A: Commercially prepared gels are denaturing gels that contain urea and SDS and use an SDS running buffer. These are not recommended because the presence of these reagents creates resolution issues with the samples being tested.
A: If bands are very dim, ensure that the imager is set to read at 705 nm and that exposure is set properly for the imaging equipment. Longer exposure of the gel may be necessary to intensify weak band signals.
A: If no bands are visible, several possibilities might exist.
- If the 80/20 Cap 1 Control Mix is visible, but the experimental sample bands are not, ensure that 12 pmol of mRNA are being used as input if using bead clean up. If using a precipitation-based clean up method, at a minimum, double the amount of input mRNA.
- If neither the 80/20 Cap 1 Control Mix, nor the experimental sample bands are visible, this indicates a reaction failure or a problem with unreactive sodium periodate. Repeat the assay from the beginning using fresh input mRNA and a fresh dilution of the sodium periodate stock.
- Double-check gel and buffer conditions and ensure that cap cores are not being run off of the bottom of the gel.
A: If several bands are visible, several possibilities might exist.
- These bands may correspond to carryover of various contaminants from the processing steps used during the in vitro transcription, capping and tailing reactions. These contaminants may consist of single or very short nucleotides that were not successfully eliminated during the clean-up step but are also labelled during the EZ-QC™ mRNA Cap 1 Efficiency Assay reaction. This is normal. The banding may differ across different mRNA samples. Do not include any of these background bands in your quantitation calculations.
- A band slightly above a Cap 0 or Cap 1 band may pertain to cap core structures that lack the N7-methyl group on the guanine cap nucleotide. This indicates an inefficiency of the capping enzyme to methylate at this site. Samples should be recapped to ensure full capping of the mRNA. Alternatively, this extra band can be used in your quantitation calculations.
