To Fizban: miRNA validation - (Feb/25/2009 )

Dear Fizban,

Did you mean you were using Taqman Low Density Array from ABI?? I would like to ask how the successful rate was for the validation by individual assays. I used the ABI taqman LDA plateform to get some interesting miRNAs and only 1/2 of them could be validated by single assays, using RNU48 and U6 as the endogenous controls. Just want to know if its my technical problem or the problem of the product design. Thank you.

Samantha



I'm using taqman miRNA panel from AB. In my experience using a card gives you a path to follow and validate. usually after a profiling i got around 10 miRNAs which seem to be interesting and they always fall down to 2/3 after validation with single assays. with latest version of miRNA arrays from AB i couldn't even be sure of what was indeed expressed. It could be due to the fact that i used a very low amount of RNA for RT. In my case RNU6B is not a good endogenous control, you should try to validate 1 or more controls for each experiment.
fizban


-Fizban-

Samantha on Feb 25 2009, 10:17 AM said:

Dear Samantha,
that's a good question! I am using ABI taqman LDA in my experiments with sometimes confusing results.
My current situation is that i do profile with ABI cards with pre amplification. I get some results, say for example 10 miRNAs that are either up or down regulated in a pateint Vs contrl comparison. usually i find that 1/3 of 10 are really regulated. Moreover i'm not always able to follow all the interesting mirnas because sometimes they are barely if detectable at all with single assays.
I must tell you that i'm using a very small amount of RNA as starting material and that's not the best condition to start bau i've had some reproducible results. by the way NONE of the internal controls such as U6 demonstrated to be really suitable for the task.
In conclusion my opinion is that ABI cards are very good if you have good and aboundant starting material and you are not totally dependent from preamplification. When using cultured cells i had very good results. the problem is that preamplification is not as linear and efficient as ABI wants us to believe. there is at least one miRNA that i didnt see at all in my samples but is really very well expressed if you try the single assay...
reality is that ABI cards are very good because they are fast and easy to use. you must be ready to lose some maybe important miRNA because of incomplete efficiency but on the other way you have a good number of mirna to work on. i have no way to screen with microarrays because i have neither the microarrays nor the amount of RNA needed so i'll stick to ABI cards and validate my screening results with single assays. i just would like ABI to stop changing miRNA primers and amplification conditions every 4/5 months
That's it, probably there's no such thing as THE method to investigate miRNAs but we all have to cope with the limits of the system we're studying. if you need help just write me a PM
bye
Fizban

Dear Fizban,

Thanks a lot for your detailed reply!!!! I could not send you personal messages as the function has been disabled...dont know how to solve this


i picked about 10 miRNAs to validate and only about 1/2 could be validated. I used cultured cells for LDA. Is my successful rate very low or similar to yours? This makes me really frustrated!!

For individual assays, i used 10ng RNA sample for cDNA synthesis and the ct value of U6 is about 22-25. For the LDA, I used 1ug RNA and got 18-20 for the ct of U6. Yeah, I agree with you that some of the interesting miRNAs become barely detectable when it comes to individual assays and that's a really big problem.

I also think that NONE of the internal controls such as U6 were universal to normalize all the miRNAs. I used RNU48 and U6 for the controls. In general, they show similar trends, but in a few miRNAs, after the nomalization by the two controls, they showed different patterns.

Just one thing to remind you as you did the pre-amplification. About two months ago, ABI told us the pre-amplification had been modified and they added a final step of 99.9C for 10 mins after the RT reaction to inactivate the enzyme. I am wondering if it may help you.

Samantha

Samantha on Feb 26 2009, 03:19 PM said:

Hi again

in my actual project i was able to "save" barely 1/3 of the initial signature, i think it veries with each project and wit the amount of RNA. During my very first miRNA screening we had only single assays in 3 different plates. it was long, hard and boring. we came up with 7 hypoxia regulated miRNAs but at the end we discovered that only 2 were significant and only could be followed....that was with single assays with old primers etc. but it's clear that nobody knows what to expect. a recent sceening in our lab took us nearly 4 months of intensive work and we came up with nothing at all!!
I actually use 3,4 ng of RNA for single assays, RT can be made in 5 ul which are then used on the same plate for real time adding 15ul of primer containing mix. that's how we do it now, we're trying to scale down.
I agree with you that normalization is hard, i use the median value of Cts from each single TLDA after removing false signals, normalize on it then i try to find the most stable miRNAs. it takes time and must be validated but without a good normalizer it's the only way. miR-16 is usually quite stable but not always.
Thank you for your reminder abot pre amplifcation conditions. i already knew about it and i asked ABI some explanation. it worries me a lot when a company changes a protocol without explaining why. i am still a bit worried because the answer was that "in a really very few cases there was an issue about stability of some miRNAs after pre amplification but it's nothing"....... i would't change a protocol fo nothing! who would? they must have changed for a good reason they can't tell. still it didn't help in my case.
that's it.
sorry to everybody else for our public/private chat, hope PM will be back soon
i appreciated sharing miRNA problems with you, just contact me again if you need
bye fizban

Some miRNAs might be "off" with ABI's method, please see Genome Res. 18, 610 (2008).

Functional Screens on Apr 4 2009, 06:17 AM said:

There was a littlebit of confusion last year when this issue exploded.
ABI had to re-check and change a lot of primers. now they say that everything's fine and under control.
my opinion: you can't have the whole picture with ABI cards, but if you are fine with losing part of the information it's still a good tool. ABi cards are more sensitive than microarrays while lacking part of the sanger database and info about other organisms.
In my case i have no way of accessing either deep sequencing or microarrays, and i have to cope with very low amounts of RNA.
For now i stick to ABI cards even if there's no linear link between pre-amplification/cards results and single assays. i can't handle more than 7/10 miRNA together so i'll go straight with ABI cards.
Again if you ask me if ABI cards are the best way for miRNAs i'll say NO, but sometimes they're the most affordable
bye fizban