RNA and glycogen get co-precipitated in alcohols (ethanol or isopropanol). Basically glycogen is insoluble in these alcohols. Glycogen seems only to become important when the concentration of the RNA (or DNA) is below a certain threshold and the threshold seems to be lower for isopropanol (ng/ml range, I can't find the "exact" concentration where RNA will not precipitate well anymore but I think in the old days, we looked it up in"The Maniatis" and it had "the" value). Glygogen may also help to precipitate very small oligonucleotides. However, glycogen is used because it also does not interfere too much with downstream applications of the RNA if its concentrations are not above perhaps(?) 7mg/ml.

Some tissues have high glycogen content and you will automatically co-purify RNA and glycogen and mess up the quantification. So, your increase in concentration may come from the glycogen that may itself absorb UV light. Supposedly, you see the "contamination" with glycogen at 230nm. The nanodrop manual says the A260/230 ratio may drop. I assume with glycogen there will be more absorption at 230nm.

So there are 2 potential answers: the glycogen itself is included in the RNA measurement and drives up the value or

you indeed precipitate more effectively RNA and oligonucleotides of short size  (especially when the concentration is very low). In the end the mix of both effects will result in an increase in "RNA" concentration and not all of it will be really RNA.

Hope this helps. 

Glycogen, like linear polyacrylamide, yeast tRNA, or any other RNA, aids in recovery of low-concentration RNA. We haven't observed a significant contribution of added glycogen to the absorbance at 230 nm, but you can always test this by adding glycogen to your buffer and if necessary blank the instrument using a glycogen solution.

In entire agreement with Dr. Witwer above.  Custom blanking buffers are an absolute necessity in all of this - and is a detail overlooked one too many times.  Not only adding glycogen, but, extracting "water" as the sample, and then following through the entire extraction process until what is used as the blanking buffer is exactly what the samples have endured; the only difference being that water was extracted and not biological sample. 

Better yet, would be the biological sample being extracted sans RNA and DNA: just sample matrix minus nucleic acids. Which is impossible in most real situations. So we once again, are at the grand impasse of best guess once again:  which sounds real.

I've not found glycogen to contribute any significant absorbance at 230nm (and yes, I tried just adding glycogen to water and speccing it). Acetate absorbs at around 210-220, incidentally, so if you use NaAc to precipitate nucleic acids, watch for that one. 

General idea is simply to create a sort of 'loose in-tube meshwork' of linear molecules, so that when you pellet it, it'll drag down extra RNA with it (because the RNA will get caught up on the mesh). Hence glycogen works, as does linear poly-acrylamide.

As Thomas says, it's generally better for increasing yields from low [RNA] samples (above a certain RNA concentration the RNA itself presumably does an effective job of forming a meshwork). I use it predominantly with low [RNA] cell culture preps where I don't have a lot of starting material, but honestly: it doesn't really hurt to include it all the time either. I use it at 10ug.tube-1 (2ml tubes).

Usually people are using glycoblue which is glycogen with a blue dye. This is used as and RNA co-precipitant to help visualize the extracted RNA. The amount of glycoblue that are added are very low and they should not increase the yield of you animal cells RNA extraction. The chances are that because you're using glycogen you can see more distinctly the RNA pellet and you extract more RNA as opposing to not using glycogen.

I think the question asked by you is well explained before. Just in continuation of Dr. Witwer, instead of blank the instrument with only glycogen solution, I process a blank sample parallely containing the glycogen or any carrier (Poly Acryl) and the other reagents used during the Isolation protocol of RNA. (As Trizol also contains guanidine isothiocyanate that strongly absorbs below 230 nm. In addition, Trizol contains phenol which is responsible for the strong absorption at 270 nm). Besides you may see at the end even the blank sample also has pellets. So, solubilizing the pellets of all samples and blank sample in equal volume of TE buffer/nuclease free water. Measure the OD and at the end subtract the value (concentration) of Blank sample from values obtained for each sample. So getting the accurate concentration of your RNA. 

Thank you every one, from your great explanation I will  follow up these steps to get better results @laurence stuart hall,  @Sudhanshu Abhishek , @George T Noutsios ,@John Hildyard ,@Jack M Gallup , @Kenneth W Witwer ,@Thomas Andl.

Simple answer is Glycogen acts as a carrier for RNA during RNA precipitation. The yield of RNA with glycogen when compared to precipitation without glycogen ll be high.

After precipitating RNA with glycogen do we need to get rid of glycogen from RNA before proceeding with cDNA synthesis? I have read that Glycogen might inhibit reverse transcriptase activity in concentration dependent manner, is it true? what concentration of Glycogen is good to use both for isolation of RNA/DNA and later use the nucleic acids for Real Time PCR/ multiplex PCR

Hi 

thank you about your valuable comments. Does any one know about hand made Glycogen? How can we  solve Glycogen powder? and how much we need from glycogen and solvant?

Best Regards

Hi, I am currently using the QIAGEN RnEasy kit for RNA extraction on my animal cells, but I am getting extremely low values so I was thinking of using glycogen. QIAGEN says it isn't compatible with this kit because it uses a silica membrane and so you have to buy their separate microRNA kit... is that right? If so, how should I go about RNA extraction with glycogen?

I found this protocol, does it seem okay?

https://assets.fishersci.com/TFS-Assets/LSG/manuals/MAN0013149_Glycogen_RNA_grade_UG.pdf

So normally I would harvest my cells in the lysis buffer RLT, then spin in the QIAshredder column, so you're saying that after this step, when I add 1 volume of 70% ethanol to the lysate is when I would also add glycogen? Would I also follow the follow-up steps from the glycogen protocol above? Like incubating it at -20 for an hour? Or would I just proceed with the QIAGEN protocol afterwards?

After that step in the QIAGEN protocol I would normally transfer the sample to the pink RNeasy mini spin column and centrifuge before adding Buffer RW1 spinning, and then Buffer RPE and spinning and then eluting the RNA with water.

Thank you so much for your help @Laurence Stuart Dawkins-Hall. I am relatively new to PCR, so your expertise is very much appreciated!

Laurence Stuart Dawkins-Hall

Laurence Stuart Dawkins-Hall

Laurence Stuart Dawkins-Hall

I would say use linear acrylamide instead of glycogen. Glycogen does interfere with NanoDrop and shows high absorbance at 230 which reduces the 260/230 ratio. Also there were reports of nucleic acid contamination in some glycogen batches.

Article Nucleic acid contamination of glycogen used in nucleic acid ...

Linear acrylamide does not interfere with NanoDrop measurements and does not interfere with any downstream analysis. I used it for PCR, RT-PCR, sequencing library construction ...etc and it works fine.

Molecular biology grade glycogen is an inert carrier used to increase nucleic acid recovery from alcohol precipitation. It is a preferred co-precipitant for solutions containing oligonucleotides or low concentrations of DNA or RNA. It will not affect the OD260/280 ratio or determination of nucleic acid concentration.

Hope that helps

Masuma

what is the concentration of glycogen we need to add?

@Yisehak Tsegaye Redda

Use between 15 - 20 ug of linear acrylamide instead of glycogen

https://www.thermofisher.com/order/catalog/product/AM9520#/AM9520

best of luck