miRNA from serum - (May/06/2011 )

Hello everyone
This is my first post so hopefully, I am posting it at the right place.
I am working on serum miRNAs. Ive few questions and I shall be very thankful if anyone could answer them for me. Since I am new to wet lab work so my questions may be very basic for you guys.

1. For each miRNA, I have 3 primers, Stem loop, Forward and Reverse. It is possible to check which of them is contaminated? Or can nanodrop detect contamination?

2. Ive been given 18s RNA to use as internal control. Well, I cant change it for now due to some reasons. I want to know is it must that ct values of 18s RNA remain same among control and infected samples? In my case, there is variation of 2-3 cycles among normal and infected samples. How can I normalize my data or make sense out of data? deltact, deltadeltact etc ??

3. I isolate total RNA with Trizol, then cDNA synthesis (20ul reaction) using Invitrogen M-MlV kit and real time using Fermentas SYBR Green (25ul reaction). In such experiments, how much the purity of RNA matters actually? If my 260/280 values are around 1.5 then such RNA samples are "ok" to proceed?

4. In the beginning, I obtained ct values for my samples in between 20 - 25 but now ct values are coming in range of 30 - 35. Total cycles I am running are 40. What could be the possible reason? is there some problem during RNA isolation? I am using 50ng RNA for cDNA synthesis (total RNA isolated from serum is around 60 - 80ng dissolved in 30ul water).

Zammy

zammy on Fri May 6 06:26:18 2011 said:

1. For each miRNA, I have 3 primers, Stem loop, Forward and Reverse. It is possible to check which of them is contaminated? Or can nanodrop detect contamination?

very geneal question. what do you mean with contaminated? have u got a problem of contamination? what makes u think so? Forgive me for being blunt but Nanodrop is just a spectrophotometer, it will give u a reading at whatever wavelenght you ask. that's it. It's useful, precise, saves a LOT of sample and time, but it's still a spectrophotometer, so you'll get absorbance, no quality data there.

2. Ive been given 18s RNA to use as internal control. Well, I cant change it for now due to some reasons. I want to know is it must that ct values of 18s RNA remain same among control and infected samples? In my case, there is variation of 2-3 cycles among normal and infected samples. How can I normalize my data or make sense out of data? deltact, deltadeltact etc ??

Again i must blunt. You are working with serum. You "have been given" 18s as internal control. Sorry, you've been given the wrong one. It's not a miRNA, it's not "internal" (you're working outside the cell, 18s will be presumably scarce and hardly stable) and it's not validate. There's no validated control (assuming that 18s is a control for everything), you must look for it and validate it experimentally. no way out. let's assume that 18s IS a good control and that it's stable also in "infected" (??) samples. 2-3 cycles means you are not using the same amount of RNA. that's it. if 18s is not a good control then you have an effect. how do u normalize? by looking for a normalizer. then u can do delta Ct and so on if you want.

3. I isolate total RNA with Trizol, then cDNA synthesis (20ul reaction) using Invitrogen M-MlV kit and real time using Fermentas SYBR Green (25ul reaction). In such experiments, how much the purity of RNA matters actually? If my 260/280 values are around 1.5 then such RNA samples are "ok" to proceed?

first of all one suggestion: you are not so sure about your methods. shift to firmer ground. gold standard for miRNA is ABI single assays. exception made for miR-33a (in my hands) they're reliable and optimized. Also: TaqMan probes are more reliable when coping with contaminations and primer dimer (it does not show). if u can compare results from different exp. using your primers and commercially available ones. Again, i use TaqMAn and not SYBR but in my experience miRNAs show well even if RNA quality is not the best (1.5 is not that good).

4. In the beginning, I obtained ct values for my samples in between 20 - 25 but now ct values are coming in range of 30 - 35. Total cycles I am running are 40. What could be the possible reason? is there some problem during RNA isolation? I am using 50ng RNA for cDNA synthesis (total RNA isolated from serum is around 60 - 80ng dissolved in 30ul water)
That's something that made me think. Ive been working with circulating miRNAs since late 2007. i'v spent months following nanodrop quantitation, i tried to improve it. then i discovered that it is not possible to quantitate them (TRIZOL extraction). i had readings but not reliable ones. i shifted to other methods (EVERY OTHER METHOD) of extraction: no way. what you'd expect with nanodrop is a peak at 270 nm. some people initially published papers with quantitation of RNA from serum and plasma. that's something i havent been hearing about since at least 2 years ago. check your readings, compare samples with similar RNA conc. for similar results and let us know. I'm trying bioanalyzer but with scarce results. that's the hard part with circulating miRNAs (assuming that cell/tissue RNA quantitated with a spectrophotometer is reliable in terms of amount and quality, mostly it is but not always). Again, let's think you RNA is what you read on nanodrop. You are doing RT with 50 NANOGRAMS! stretching the limits a littlebit, isn't it? is it primer specific RT? i assume that's your case since you wrote stem loop. in that case you are using too much RNA for single RT. Agin check RNA yield in some way. one way would be to che several miRNAs on each sample and see if everyone has the sam trend when compared to other samples. if yes: you have different amounts of RNA in your samples (or: u are very unlucky and chose all "infection" modulated miRNAs, that's rare but can happen!).

one last general consideration that is not against you but against some people way to do research and MAKE other people do it in the wrong way.
Please always ask yourself what u are doing and how. That's my opinion but i can't stand to hear people say: "I have been given a normalizer". You do research: that should mean posing a question and trying to find the right answer in the best possible way condering also that you have to think about false positive results and artifacts. before using a normalizer you MUST ask yourself if it's suitable. read literature, understand it and try also to understand if literature on the argument is reliable. circulating miRNAs are fascinating but we know VERY little. one thing that is almost certain is that usual housekeeping genes DO NOT APPLY. you can find people accepting them because of habit. (yes i said it: habit. how many reviewers ask about normalization and UNDERSTAND how normalization is made?). Lately spike in normalization is the accepted standard in circulating miRNAs: if u look well you'll see that in many cases normalization is done the wrong way. In conclusion: u want to do research? good but do it and don't let anyone feed reserch to u without thinking what u have been given.
bye
fiz