Dear Clay

• Yes a potential disadvantage of glycogen is that is does absorb at 230nm, thus reducing your 260/230 ratio
• There has been much debate on this topic including by participants on RG
• However, in my experience unless your 260/230nM is < 1.0 simply ignore the peak:
• Glycogen as a carrier is designed to be inert so will not interfere in any way
• What is your 260/230 ratio incidentally ?
• If > 1.0 then simply ignore that peak; It will not interfere
• If < 1.0 and especially if ~ 0.5 you might have additional concomitant issues with guanidinium salts and/or Trizol : If present, putatively indicated by very low 260/230 ratios these can inhibit RT
• In these cases either re precipitate or add an equal volume of 70% ethanol to your precipitated and re suspended pellet and column purify
• In terms of potential Trizol and/or GITC contamination that might augment your 230nM signal caused by Glycogen I would precipitate your RNA; Remove supernatant; add in 0.5 to 1ml of ROOM TEMP 70% ethanol and then vortex to dislodge pellet; spin and repeat:
• In my experience, this 2 x room temp 70% ethanol wash removes Trizol and/or GITC contamination  

Do you have similar data from a negative control reaction? if not, repeating the experiment without template with and without glycogen and comparing the results to your positives should  allow you to specifically identify if glycogen is the cause. That said, I am familiar with research identifying glycogen as a source of DNA contamination, and it seems reasonable that it may carry other contaminants as well, especially if the prep is poor. make sure you are using molecular (or RNA) grade glycogen and always, always, run control reactions.

Dear Clay

• Yes a potential disadvantage of glycogen is that is does absorb at 230nm, thus reducing your 260/230 ratio
• There has been much debate on this topic including by participants on RG
• However, in my experience unless your 260/230nM is < 1.0 simply ignore the peak:
• Glycogen as a carrier is designed to be inert so will not interfere in any way
• What is your 260/230 ratio incidentally ?
• If > 1.0 then simply ignore that peak; It will not interfere
• If < 1.0 and especially if ~ 0.5 you might have additional concomitant issues with guanidinium salts and/or Trizol : If present, putatively indicated by very low 260/230 ratios these can inhibit RT
• In these cases either re precipitate or add an equal volume of 70% ethanol to your precipitated and re suspended pellet and column purify
• In terms of potential Trizol and/or GITC contamination that might augment your 230nM signal caused by Glycogen I would precipitate your RNA; Remove supernatant; add in 0.5 to 1ml of ROOM TEMP 70% ethanol and then vortex to dislodge pellet; spin and repeat:
• In my experience, this 2 x room temp 70% ethanol wash removes Trizol and/or GITC contamination  

Doing a negative control prep is a great idea, I will do that next time.

My 260/230 ratios for the glycogen-containing samples in my last set were 0.8, 0.9 and 1.2. In general they tend to hover around 0.8-1.1. The 260/230 for my glycogen-free samples are all around 1.9. So it seems like if I have any contamination it must be pretty mild.

I have been washing my pellets with ethanol at least twice, but I have never vortexed and spun them down before. That might help to get rid of the last bit of contamination, so I will definitely give that a shot.

Thank you both so much for the help!

Dear Clay

I will emphasise one or two more things:

• Vortex your pellet in 1ml of ROOM TEMP 70% ethanol if you are not currently doing that
• To state the obvious solubilisation of salts occurs more efficiently in a solution at room temp than 70% ethanol stored at -20C
• The rational for cold ethanol is that is general terms it is better to keep RNA cold - Generally true - but in this particular context precipitated RNA is relatively stable for short periods (in aqueous solution that is NOT true) and so the benefits of extra removal of salts and possibly trizol by utilising room temp alcohol on balance are for the best
• Also, having spun your pellet for 5 minutes, following 2 x ethanol washes, remove most of the ethanol then spin again for 1 min to bring the residual ethanol on the sides of the tube down and remove this using a p10 tip
• This will most efficiently remove salt laden alcohol and also has the added benefit that drying of you RNA pellet can be achieved by leaving on ice for 10min followed by 10min at room temp
• Regarding depressed 260/230 ratio due to glycogen - which will not affect downstream processes  - and GITC/Trizol that might - both appear the same by Nanodrop profile but will definitely look different on an Agilent Biochip if you have recourse to RNA analysis using that method:
• If samples contain deleterious contaminants then a small peak in front of the 2 x main ribosomal rRNA peaks will appear on the trace ( co localising with the 5srRNA peak in fact)
• Re purification will remove this peak and result in a 260/230 ratio > 1.0
• In my experience this re purification converts refractory samples into ones with down stream utility
• In contrast samples contaminated with glycogen might evince a 260/230 ratio < 1.0 but will show no extra peak up stream from the dominant rRNA peaks; and will incidentally be fir for purpose  

I recommend you to reprecipitate your RNA by using Na-Acetate and ethanol then measure the values again.. this will correct your 260/230 ratio. prepare AN acetate using DEPC water.. this method can help you alot to save your samples.