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RPC Optimization Guide v1.0.2

Introduction

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:


General

Resuspending the RPC

  • Thoroughly clean the work area with 10% bleach, and again with 70% ethanol.
  • Use a fresh pair of gloves for this step and discard them immediately after.
  • 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.
  • Make sure to thoroughly clean the work area again by using 10% bleach, and again with 70% ethanol.

 

Cleaning

  • 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 easily contaminate an entire 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 may encounter the RPC Buffer because bleach will completely degrade the RNA. It is good practice to clean the work surfaces or tools with bleach to decontaminate, then clean over the area or tools again with 70% ethanol to clean up any bleach residue.

 

Storage

Proper storage of the RPC Buffer will prevent early degradation of the RPC or misuse post-expiration.

 

While In-Use

  • While working with the RPC, keep it cold using a cold block or on crushed ice until it can be returned to 4°C storage.

 

Short-Term Storage

  • The RPC should be stored 4°C for up to one week from the date of resuspension.
  • If 4°C storage is not available, the RPC buffer can be kept at room temperature for up to one week but may degrade faster than expected. It should be moved to 4°C storage as soon as possible.


Long-Term Storage

  • Always freeze the buffer as close to the date of resuspension as possible.
  • If the RPC must be stored for longer than one week, the buffer can be stored at -20°C.
  • The buffer must be used within 3 days from the first freeze/thaw (F/T) cycle, unless frozen again.
  • The buffer may be frozen again, for a maximum of 3 F/T cycles.
  • If it will not be frozen again, it should be stored at 4°C until it expires.


1. Freezer Options

  • The best option is a lab-grade freezer that has advanced temperature control and should not experience severe changes in humidity.
  • If there is no lab-grade freezer available, a common residential fridge/freezer combination can be used to store the RPC. The temperature is typically around -18°C in these freezers.
  • If using a common freezer, always place the tube in a freezer-safe zip-top bag to protect the buffer from changes in humidity.

2. Aliquot

  • If part of the buffer needs to be stored long-term, the resuspended buffer can be aliquoted out into the desired volume plus 10% more.
  • The aliquots should be in screw-cap tubes which are preferable to snap-top tubes, such as Eppendorf tubes.
  • It is best practice to aliquot the buffer immediately after resuspension so it may be placed in the -20°C as soon as possible.

3. Thawing

  • The buffer should be completely thawed before using to ensure the concentration is correct.
  • The buffer can be thawed by placing into 4°C, room temperature, or warming the buffer between gloved hands. Do not apply the heat of any other form to speed up the thawing process.
  • The buffer is viable for up to 3 days after the first thaw and must be discarded if unused after this time.
  • If it will not be used within the first 3 days after the first thaw, it can be frozen again if the 3 days' time has not expired. If freezing the RPC again, it is best to return it to -20°C storage as soon as possible.
  • The RPC can go through a maximum of 3 freeze/thaw (F/T) cycles, after which it must be discarded no matter the amount of time that has passed.

 

Workflow

Paying careful attention to key points in the workflow will minimize RPC-related failures.


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

  • 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, and 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.



If you have questions about the topics mentioned here, or anything else, please contact the Customer Success team at support@biomeme.com.



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