Specialized and Automated RNA Isolation Support—Troubleshooting

• Precipitation of miRNA: Follow the instructions for the mirVANA™ miRNA Isolation Kit or the mirVANA™ PARIS™ RNA and Native Protein Purification Kit.
• Double phenol/guanidium (chaotropic reagent) extraction: Disrupt tissue with phenol/guanidine, then extract with phenol, centrifuge, remove the supernatant, and precipitate the RNA.
• Glass-fiber filter: Disrupt the tissue in a chaotropic agent, bind the RNA to a glass-fiber filter, then wash off unbound DNA/protein/salt/nucleotides and elute the RNA.

You would have to separate the virus from the cells prior to isolation to avoid contamination of the sample with cellular nucleic acids.

Low RNA content or a loss of the pellet during ethanol precipitation could lead to a low RNA yield. Additionally, various tissues have different RNA content, and the yield is dependent on the sample.

There are several possibilities:

• Too much total RNA may have been used: Please check the amount of total RNA you are using (we recommend between 2 and 10 µg total RNA).
• A low amount of magnetic beads or probe was used: Use the recommended amount of probe or beads.
• Improper handling or drying of the beads may have occurred: Follow the recommended guidelines for washing/mixing beads; do not allow the beads to dry.

Perform a DNase I digestion with the total RNA sample to remove any genomic DNA contamination before isolation of RNA using the RiboMinus™ kit.

Wipe the magnetic rods with a soft cloth or tissue paper soaked in a mild detergent solution, soap solution, or alcohol.

The protocol will not start without the tip combs inserted into the tip comb holder.

If the starting material is too viscous, the magnetic rods will not be able to collect the particles. Dilute the sample and check that the sample is properly homogenized and lysed.

This happens sometimes, but it will not affect the yield because the sample has been released from the particles.

A potential cause is that the plastic tip comb has warped slightly due to its design. This is why they are packed in pairs, so that they maintain their shape. Flex the backbone of the tip comb and try again.

Turn the machine off, and inspect it for any visible damage. Make sure the correct head is being used for the protocol being run. If the machine shows no visible damage, turn the machine back on. This should reset the machine. If the problem immediately occurs again, turn the machine off and gently move the magnetic head apparatus to the center of its travel path, then turn the machine on again. If the problem persists, the head may need to be realigned by a service call.

Unfortunately, freezing of the RNA binding beads will render them nonfunctional. Please discard them.

Please follow these recommended steps:

1. Press the ESC key on the keypad to return off the main screen.
   
2. Select 1 to enter the manual screen.
   
3. Select 2 to return the tips to the holder. The instrument will also move all axes to the original position.
   
4. Turn off the machine, remove the card, reinsert the card, restart the instrument, and run the protocol without reagents present.
   
   Should the same error code still appear, try the following:
   
   Turn off the machine, remove the card, reinsert the card, restart the instrument, and under the manual menu, choose option 2, return tips. Then run the protocol without reagents.

Wipe the magnetic rods with a soft cloth or tissue paper soaked in a mild detergent solution, soap solution, or alcohol.

The protocol will not start without the tip combs inserted into the tip comb holder.

If the starting material is too viscous, the magnetic rods will not be able to collect the particles. Dilute the sample and check that the sample is properly homogenized/lysed. Adding low amounts of detergent will improve the collection of magnetic particles as well. Quickly centrifuge the plate to sediment the particles to the bottom of the plate.

This happens sometimes but it will not affect the yield because the sample has been released from the particles.

No, once a protocol was stopped, it will restart from the beginning. The extraction procedure can either be finished manually by using a magnetic stand or a modified version of the original KingFisher™ Flex protocol would have to be created that begins at the point where the run was stopped.