Not enough cDNA for qRT-PCR - how to increase cDNA yield or save cDNA? - (Feb/20/2006 )
I am trying to optimise an assay for a gene in reptile muscle tissue. Its tricky to get RNA out of the tissue, and so I often have very low, sometimes unusable concentrations of RNA ( I use tri-reagent - I also do extractions at the same time with other tissue and they work fine with high concentrations).
After RT, I have about 40 ul of cDNA to use from each extraction, if the extraction worked and the RNA is not degraded (maybe half the time or less it seems ok!). I use 2 ul per PCR reaction.
As I am trying to optimise my PCR assays with several genes (3 test ones and a housekeeping), I need lots of cDNA for test runs. I keep running out of cDNA just in testing the MgCl concentrations, primer concentrations, DNA concentrations and temperature, before I am able to run a standard curve etc.
It is very time consuming to do new RNA extractions constantly, and I am using up all my tissue!
Does anyone have any ideas of a method to amplify cDNA, or some other way to improve or preserve my cDNA yield?
the RT reaction is all about dependency on template; I suspect you need to beef up your RNA yield and the cDNA will take care of itself
especially if you are getting inconsistent yields, I would suspect degradation may be a culprit.. some suggestions would be (if you are not already doing all this; if you know this stuff I apologize)...
turn your RNA into cDNA as quickly as you possibly can; cDNA is much more stable
check your RNAse-free-ness of reagents, pipets, bench, EVERYTHING
turn your tissue into RNA as quickly as possible (RNAlater is good stuff, but still I feel that sooner is best with this step). also, have you tried different tissue homogenization (did I get this word right? I haven't had my coffe yet) methods?
visit some sites and look for tips and stuff regarding working with RNA; even if you've read most of it before, I bet there are some gems in there that might help you find the source of the problem
lastly, have you considered assays-on-demand? I don't use them myself, but I could see how the cost of your initial sample prep and optimization could get out hand, and perhaps the expense would be worth it
you've come to the rescue again! This forum is so useful.
Yes, those are all good ideas. I will look into it again. I think turning all my RNA into cDNA straight away, as soon as I have a sample with a decent yield and quality, would help.
What are 'assays on demand'?
I think there are multiple companies that produce them, but go to ABI's website and type in 'assays on demand' as a search term and you will see what I am talking about
you get a primer/probe set from them that has already been wined, dined, and troubleshot for you, guaranteed to work the first time you run the plate if you follow their protocol
anyways, something worth looking into
I've been using the assays on demand, and also I did my own relatively efficiency test with two of their genes and a housekeeper. The test passed just fine, I got good amplification of my samples, and the amount of work put into making the primer mixes was cut more than in half. I have to vouch for them!
Back to RNA handling...Aimikins, I was taught that leaving RNA precipitating in isopropyl alchohol overnight at -20°C improves yield, so that is what I've been doing. However, that doesn't really follow the "handle RNA quickly" rules that other people here seem to follow. Do you (anyone?) have an opinion on that? Thanks!
I do not know of that method; at which step do you perform the precipitation?
I use a column-method, with the increased-yield steps added (I use the smallest amount of tissue cells possible so I like to get all the RNA I can)
I elute into a fairly large volume. At this point, I assume that the only source of contamination is, well, me. I then ETOH/oAc ppt overnight at -80 to concentrate it down. as soon as the pellets are dry, I do the RT step
I do not hesitate to ppt the RNA overnight, because the RNAse and stuff from the cells should be gone by then? and I would think the alcohol would inhibit it anyways (since you don't have to use nuclease-free ethanol), although I would not store it like that for longer periods. My feeling on processing quickly...hmmm....basically, I try to harvest the cells as quickly as possible, then prep the RNA right away. I also don't store the RNA very long (maybe a day or two) before turning it into cDNA. I have stored it longer at times with no particular ill effects, but I just feel like it takes so much work to get to the damned RNA, long before you even get to the qPCR, that paranoia about RNase is a good thing
what do others think?
on that issue of getting a better RNA yield with extended periods of ethanol precipitation, has anyone tried precipitation with 12 M LiCl?
I just read a protocol that recommended this for better RNA yields. I think its done as an extra step, after dissolving the purified RNA pellet.
Thanks for your reply, Aimikins. I use the TRIzol method, and the isopropyl is added for ppting the RNA after the aqueous phase is separated. I'm glad to know you don't have trouble keeping RNA in alchohol overnight, and RNA should be more stable as a precipitate anyway. Thanks again!
I do not think that it is necessary to ppt it overnight; usually that just works out well with the time flow of the experiment. It is quite likely overkill, I would guess that an hour or two would be plenty sufficient
I have not tried LiCl, but I too have heard that it works well