The RNA Process Control (RPC) is an internal positive control designed to act as a process control for every sample tested. Biomeme RPC contains MS2 bacteriophage which is the target for amplification. Possible RPC-related issues include late Cq values, lab contamination with RPC, early degradation due to RPC stock contamination, and degradation past storage recommendation date.
Here are some tips to follow when working with the RPC:
Paying attention to certain key points in the workflow will minimize RPC-related issues.
Mix the RPC Before Use
Biomeme recommends using a Vortex to mix the RPC for 15 seconds before use. If there is no Vortex, shake the RPC bottle vigorously for at least 1 minute.
After the initial resuspension of the RPC, the MS2 will begin to settle within the RPC bottle. It must be mixed thoroughly before use, otherwise, the concentration of RPC buffer added to the M1 Sample Prep Cartridge will be less than desired, leading to late Cq values and/or invalid results.
Inspect the pipette tip for liquid while pipetting 20 μL of RPC
It is good practice to do a quick visual inspection of the pipette tip when pulling up liquids. This is especially important if using the 20 μL Fixed Volume Pipette.
Dispensing RPC into the M1 Sample Prep Cartridge
When adding the RPC to the M1 Sample Prep cartridge, place the pipette tip to the bottom of the 1st chamber at a slight angle, and then release the RPC, making sure not to bring any liquid back out.
Because 20 μL is a small volume, using this method will ensure that the RPC is being added to the liquid, and not accidentally forming a droplet elsewhere in that chamber or on the inside of the foil.
Extracted Sample Degradation
RNA is highly unstable, even more so once it is extracted and purified out from its natural environment. The extracted sample is good for up to 4 hours but should be used as soon as possible after extraction.
If the extracted sample will not be run within 15 minutes post-extraction, keep the sample on ice until it is ready to be loaded into the Go-Strip.
Loading the Go-Strips
When adding the extracted sample to the lyophilized pellet in the well of the Go-Strip, make sure to pipette up and down at least 10 times to thoroughly resuspend the pellet. Insufficient mixing at this stage may cause late Cq values and other non-RPC-related issues.
Do not mix the extracted sample into the Go-Strip ahead of time. Working one run at a time, once the samples are loaded into the Go-Strips, immediately place them into the thermocycler and start the run.
Proper storage techniques will prevent early degradation of the RPC or use post-expiration.
Resuspending the RPC
Use a fresh pair of gloves for this step and discard them immediately after this step.
Mark the date of resuspension and the date of expiration directly on the RPC bottle.
Store the RPC at 4°C, and use it within 1 week.
Thoroughly clean the workspace before and after resuspending the RPC. If possible, designate a section or table in the lab for RPC resuspension since MS2 can very easily contaminate a lab.
Keeping the lab and the work area clean prevents nucleases from getting into the bottle of RPC. Nucleases are enzymes that are present in the environment and will degrade the RNA if it gets into the RPC Buffer.
Be careful when using bleach to clean anything that will come in contact with the RPC Buffer because bleach will degrade the RNA. It is good practice to clean the work surfaces or tools with bleach to decontaminate, then clean the area again with 70% ethanol to pick up any bleach residue.
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