Mistakes with reverse transcription - Can you tell me the impact? (Jan/13/2006 )
I reverse transcribed a large number of samples today (96), and I meant to include 8 no amplification controls. I made the stupid mistake of adding my enzyme to my RT master mix before creating the amplification controls (I just automatically went down the list of things to add); however, my volume of reverse transcriptase was calculated NOT to include those 8 NACs. I did not realize this until after I completed the RT reaction. So, now I have no NAC's and the amount of enzyme would have been slightly low for all reactions.
In addition, I also figured out too late that my pipette was set incorrectly, and I pipetted less than 100 µL/well (probably around 90 to 95 µL).
Do you think these 96 samples are all lost? Or are these mistakes small enough that I can procede with PCR?
.......Maybe I should not do experiments on Friday the 13th. I knew I should have stayed home today after I lost a favorite earring down the drain this morning.....
P.S. You can add me to the "stupid lab girl" list in the Lab Joke forum...
I would guess a little less RT is not a problem, as is a little less sample/well...the only problem is if your buffers and such are at different concentrations than they should be?
90 or 95 ul/well shouldn't matter; the ROX will cover you for small differences in the amount of mix added to each well across replicates (I assume on a good day there is a difference of a couple microliters anyways; pipettes and humans are not perfect, that is what the passive reference is for)...I would also think a little less RT is OK. I think most companies tell you to use more enzyme than you need to and less should be OK if you didnt' skimp on reaction time
it is only the buffering conditions and template quantities that should be the most important. if those are off, I would repeat it 
how did you do it? and if you did perform the reaction, how did it work?
A
In many commercial protocols, there is a 10-fold excess of reverse transcriptase in the reaction.
Since RT is known to inhibit Taq, having a little less than normal in the reaction will probably not have a negative effect on your final results.
Thanks for your replies! I'm glad to know that my samples might be ok. It is so frustrating to calculate everything so carefully, and then be off by a little bit when actually doing the experiment.
Aimikins, all the other reagents were added at correct concentrations. This is normally what I do (please keep in mind that I have not been trained and have had to teach myself RT-PCR, so feel free to criticize!):
1. I add everything into my master mix except the RT. I then create my NAC wells by adding the correct amount of MM-RT. Next, I add RT to the [Master Mix-(volume used for NACs).
2. In this experiment, I chose to convert all the RNA I had into cDNA in 2 µg RNA/well reactions (I wanted a large cDNA pool from which to optimize). So, I measured the [RNA] at A260, and then took the full volume of RNA for one sample + DEPC H2O + Master Mix such that the final concentration of RNA is 20 ng/µL. I then add 100 µL of this large mix into, say 10-12 wells, with 2 µg RNA/well.
3. I do this for all samples, and then use the RT reaction conditions for the ABI 7300.
As for as the rest of the buffers - I can't wrap my brain around whether those are at correct concentrations or not. Since I added my RT too soon, the final volume was not as I had originally planned, right? I mean, by definition of adding less RT than planned, are ALL buffer concentrations are ultimately off? Sorry - I confuse myself sometimes!
Oh, and I plan to run my qPCR experiments on Monday, but I have still been unable to optimize my system. We just got the Agilent 2100 Bioanalyzer, and I figured out that the RNA I had been working with previously was very degraded (yikes). I just made this huge new batch of RNA to start again with the optimization experiments - so I suppose if the efficiencies are good it's fine, but if they are not, I will have to add a poor RT reaction into the pool of possible reasons.
I do my RT's in little eppy tubes separately in a heat-block; so my set-up is different
how much does the RNA you add contribute to the total volume in each well?
if the RNA contributes only a little to the volume you should be OK; if the RNA contributes a lot to the volume you might be off
If I were you, I would qPCR like a couple of your cDNA samples, perhaps a couple controls where you know more or less what to expect, and see what you get...I would not run all the samples if there is doubt about your cDNA. but that is me...I like to roll the dice and see if it's going to be OK. I do think many enzymes are perhaps a bit more flexible than we are initially led to believe...so for this experiment I would bet you can still get usable data, but if you want to compare, say, this batch of results against another batch there may be some inconsistencies. that is the only real problem that I foresee. if you are still setting up your efficiencies and such I don't imagine the overall impact will be too huge but it's hard to say till you run the samples 
I add enough RNA to get a total 20 ng/µL in each well. So, in the past experiment, I added about 3-5 µL RNA/100 µL rxn. Hopefully that volume is small enough that it is ok. I will definitely run a small qPCR experiment on Monday just test test my cDNA and I will let you know how it goes!
Just a follow-up to let you know that I've run 2 plate with this cDNA, and although my efficiencies aren't great (about 80% - and I had that problem before this), the cDNA seems to work fine. In fact, I am getting tighter results with my duplicates and lower Ct's with higher dilutions this time. Thanks again for all the advice!