That is completely bizarre !

I would re treat fresh aliquots of RNA with a new aliquot of RNAse

I would also recalibrate/check your qubit device

I generally found them very reliable when I used ~ 10 years ago

Also, check the age of your fluorescent qubit dye and make sure it is no more than 1 year old and has not been freeze thawed too many times

If in doubt purchase more

Have you considered including a control sample in the treatment process? This could help to tell if it is something to do with the digestion or just the effect of temperature and time on the sample.

First of all, what are you trying to achieve by digesting the extracted RNA? Pure DNA samples?

1. Technically Qubit kits are not meant to measure digested RNA simply by the way they work. Qubit measures fluorescence. The staining agent in RNA kit interacts with uracil residues producing linear fluorescence, more uracil more fluorescence. RNAse does digest the RNA, but it doesn't remove the uracil from the sample and it still interact with the staining kit agent producing false data. If anything digested RNA would have free uracil that has better interaction with the staining agent explaining the increased readings.

2. Use Nanodrop, it measures the RNA absorbance, digested RNA produces a completely different absorbance profile than undigested RNA.

3. Try to remove the RNA by different method i.e. using RNA Extraction kit, but instead of discarding the flow throw at the first step extract the DNA from it, RNA stays in the column and DNA in the flow-through and then check your DNA/RNA values with the nanodrop.

4. The issue in the leaf samples itself with something that was extracted along with the nucleic acid that interacts with the Qubit RNA kit producing false readings, again try Nanodrop instead of Qubit.

4. If you want more information try to run your digested and non digested samples on an agarose gel to see what is going on in your samples after RNAse application. If you see that 18S and 28S ribosomal RNA bands have disappeared turning into the faint smudge and you have a clear DNA band that you are successful in digestion. You can also evaluate your DNA degradation by observing fragmentation bands.

5. Actually you can simply extract the DNA band from the gel ensuring the elimination of the RNA even without digestion and then measure the DNA.

I hope it helps, there are many approaches to solve your issue, and I would start with Nanodrop.

Hi Anton Lennikov . Thank you for your insightful response! We actually have done column DNA purification on separate aliquots of these samples. We are including a crude extraction protocol employed by several other research groups to eventually compare quantitative measurements of a contained pathogen in both extract types. Our pathogen quantification will be DNA specific but normalizing the data will require knowledge of input concentrations of both ss and dsDNA. My goal was to eliminate the RNA--as the dye in the Qubit kit interacts with it as well--without deviating too much from the crude extraction protocol. I've ordered a synthetic copy of our pathogen genome and will use a standard curve to relatively quantify my pathogen to overcome the limitation of the Qubit chemistry.

I would suggest using nanodrop measurements. It easilly destinguish intact and digested DNA/RNA as it measures direct absorbance, rather than a dye.