Dear Pal,

It is probably an aerosol contamination, I have experience with miRNA we have performed ddPCR as well. I also initially faced contamination issues while experimenting. I struggled to solve the issue of contamination. I try finding literature to solve the issue. The contamination can be avoided through the de-contamination solvent mostly for RNA work we can use RNaseZap™ RNase Decontamination Solution. One of the best solutions I have used to mitigate the contamination. Sometimes we used to perform the extraction under a controlled environment. Still, if you are facing the issues that must be: 1. Your extraction reagents might be contaminated. 2. If you elute the RNA at the last step that time if you are using kit buffer it is okay, most of the labs will elute with Nuclese-free water, wherein we must ensure before eluting. Sometimes the RNA can be on the instruments too. So, you wipe the alcohol at the place where you are placing your sample. I used to change the gloves while performing RNA extraction while eluting I used to change with fresh ones. After procuring the RNaseZap, I am just using the solution to clean at the last step. 3. I expect your biomarkers to be specific to mIR-16p and 21p. Sometimes this also goes bad if you dissolve/suspend the primer with contaminated water. You ensure the primers are reconstituted with fresh nuclease-free water or contaminated-free elution buffer. 4. cDNA conversion if you are performing you ensure the safest place to add your RNA to the cDNA mix. While Quantifying your RNA, you must ensure while opening the tube you should be careful with environment RNases. 5. Clean your pipettes with alcohol before and after performing the experiments. I think you are performing Good laboratory practice I think this is not a serious issue in your case. 6. Ensure your PCR mix is free from your target miRNA. you can check by performing this combination:*

1. PCR MIX+ PRIMERS - NO water

2. PCR MIX+ PRIMERS + water ( like NC)

3. Fresh vial Master Mix and other chemicals are common

You can try combinations like this! If you still facing the issue shut the work for a day perform fumigation and restart all your experiments. Hope this information is precise and it will work for you. All the best for your future experiments. Good Luck!

Use a fresh pair of gloves and close all the PCR tubes from a fresh bag. Often contamination can be spread when trying to pick open tubes from a bag.

Dear Dr. Pal

It gets frustrating when one faces contamination during PCR. The unexpected signal could be caused by contamination of reagents with template, DNA contamination or amplicon contamination.

I have listed down a few measures which you could adopt to prevent contamination.

1. Use separate rooms, one to prepare the PCR master mix and the other to add the template. This will help prevent introduction of template into the reagents. As far as possible, use a benchtop PCR hood to prepare the master mix as this will help minimize the risk of contamination, and a benchtop hood is simple to decontaminate.

2. Try to keep the components of the PCR mix like the enzyme, primers and probes, nuclease free water as well as the consumables like the pipettes, microfuge tubes and tips in a room that is not used to isolate or store the template.

3. Pipettes are a common source of contamination by means of aerosols. So, make use of filter tips that will act as a barrier between the pipette and the liquid being measured, thus preventing the transfer of aerosols into reagents and samples. Also, make use of positive displacement pipettes that will help limit the risk of aerosol contamination.

4. Clean pipettes and the working surface area with 5% bleach. You may also use UV sterilization to decontaminate the equipment including the pipettes and the tube racks.

5. Use a no ‘RT’ control in your experiment by omitting reverse transcriptase in the reverse transcription step. This will help you to identify genomic DNA contamination in RNA preparation.

6. Try to store your stock solutions in aliquots for one time use to minimize the risk of contamination of the stock solutions. Also, if you observe contamination, you may always repeat the experiment with fresh aliquots.

Since you have observed contamination, my advice to you is to repeat the experiment with fresh stocks. You may have to discard the existing reagents.

Regards,

Malcolm Nobre