OK I finally did that experiment that people asked for … and more.
If you want to know why I performed this experiment, read this post on RNA treatments for autism. Here I present to you evidence that your body is secreting enzymes, called RNAses, that will chew up your RNA in minutes.
So the experiment:
500ng of RNA was mixed with either
– control buffer (100mM KCl, 10mM Tris buffer pH7.4)
– 1ul of DNAse (1mg/ml), this is what you would call a negative control – no degradation should be seen here
– 1ul of water applied to sweaty skin
(After working a bit with my latex gloves, I puled off my gloves and applied 20ul of buffer for 5 secs and then collected the drop of sweat+buffer sample)
– 1ul of saliva (collected from a baymate)
– a single strand of hair (again collected from my trusty baymate)
– a 5mm long toe nail shaving (again from my baymate)
– 1ul of RNAse (1mg/ml), this is a positive control – we should see plenty of RNA degradation here
(note ng = nanogram; ul = microliter = 1/1000 of a milliliter)
All samples were brought up to 5ul total volume using the control buffer and incubated at 37 degrees Celsius for 5min. Note that the RNA is present at 0.1mg/ml, a decent concentration.
The reaction was terminated by adding 5ul of 50% formamide loading buffer and then incubating the samples at 90 degrees Celsius for 10min.
Samples were loaded on a prestained non-denaturing agarose gel (I didn’t have time to make radioactive RNA and pour a denaturing gel) and a 90mA current was applied to the sample for 30min.
And the result:
The RNA migrated from top to bottom. The longest RNA strands migrated the slowest and are thus on the top of the gel. Smaller RNA strands migrated faster are thus on the bottom. To show you how fast the bands migrated, I loaded molecular weight standards in the first lane. 2kb = 2 kilobases = an RNA strand that is 2000 bases long. The substrate for this reaction was an RNA species that I synthesized in the lab. This transcript is 500 base pairs long. It was capped at the 5′ end and polyadenylated (i.e. a poly-A tail was added) to the 3′ end. There is about 200-300 adenines in the poly-A tail and so the whole RNA is about 700 to 800 bases long. This is a good mimic for the majority of the mRNA inside a cell (actually this particular transcript is fairly stable compared to the typical mRNA transcript). Although the samples were loaded on a non-denaturing gel, this RNA tends to run at the proper molecular weight as it does not form much secondary structure.
As you can see,
– DNAse had little effect, the RNA is still there and migrates at the right speed.
– Sweat can degrade RNA. So despite the fact that this reaction was carried out with water that came into contact to a sweaty hand, the end result was that the RNA was effectively chopped into bits after a simple 5 min incubation. The size of the bits are about 10-100 bases long.
– Saliva can degrade RNA very effectively. Don’t forget that this sample’s saliva is 5 times less concentrated than what is in your mouth. Some small RNA bits survived the treatment (maybe 10-50 bases long) but the majority of the RNA has been decomposed to smaller stuff (single nucleotides) that can’t be stained.
– The single strand of hair had minor effects. Actually I quantitated the intensity of the band and it’s only 80% of the control – so as much as 20% of the RNA was affected, but the majority of the RNA survived.
– Toe nail clippings were deadly to the RNA. Lesson: don’t try to salvage RNA that landed on your toe nail!
– pure RNAse was … well lets say that I added the equivalent of a nuclear bomb to the poor RNA. Not even a trace survived.
So there you have it. We are all oozing RNAse, the enzyme that degrades RNA. Stick RNA in your kid’s mouth and it’ll be gone – digested by the RNAse found in human saliva. And there’s a whole lot more RNAse in your stomach – I promise you that no RNA will ever reach your intestines (where all the nutrient absorption takes place). If you read my previous post on RNA & autism, you’ll find out that “therapeutic” RNA is sold in lan aqueous solution with a dripper. Even touching the dripper your bare hands is enough to degrade all the RNA inside the vial in minutes. The fact that your body is full of RNAse is not surprising as our bodily secretions help protect us from many viruses that have RNA genomes.
Direct RNA ingestion will never work.
I would like to thank baymate for suplying bodily fluids and parts.