RNA concentration and quality - How to be sure of RNA quality? (Apr/27/2007 )

I will be doing microarray in near future. so now i need to extract good quality RNA from epithelial cell line for that.
After i extracted the RNA, i used 3 methods to check the quality of my RNA:
1. Run an electrophoresis gel
2. Nanodrop
3. Bioanalyzer

Electrophoresis gel gives me 2 nice bands. But Nanodrop tells me my RNA purity is poor (A260/A280 only 1.7), however the concentration is high (>4000ng/ul). After running the Bioanalyzer, Bioanalyzer couldn't even give me RIN no, and tells me my RNA is not good at all. And also my RNA concentration is only 2000ng/ul (half of what Nanodrop gives me). I used 2 Nanodrops machine in different lab, both give similar concentration. but the reading Bioanalyzer gives is half of Nanodrops reading. My labmates also face the same problem.

Which instrument should I trust? Nanodrop or Bioanalyzer? it seems they give different reading (concentration)?
Do anyone face the same problem like i do?

Is the type of cells (such as lung cells, spleen cells) matter? cause some of my friend extracting spleen cells RNA all facing the problem of high background on electrophoresis gel.

Can you do a simple RNase digestion or DNase digestion?

Absorbance wouldnt really distinguish both DNA and RNA samples. just my 2 cents worth of opinion.

Hi,

This is a common mistake.... Your RNA is probably fine. If you read the instruction manuals for both the nanodrop and bioanalyzer you will find that your RNA is way too concentrated for it to measure properly. The bioanalyzer works best when you add approximately 500ng only the chip. The nanodrop can only handle approx 2500ng/ul from memory but the high ranges are not as accurate as the middle ranges. I would do at least a 1:10 dilution of your RNA then you will be able to trust both pieces of equipment. By the way, I do trust the nanodrop more for obtaining the concentrations of the RNA than the bioanalyzer. I am just old fashioned though. Bioanalyzer is great for showing you the quality of the RNA.

Cheers
Ant

Yeah I tried the Qubit once. It also gave me different results. BUt again, the RNA has to be pretty dilute for measurement in Qubit (max 100ng i think). Like I said, I use the spectrophotometric measurments as I am old and have used it for years even though it is more likely to measure all the nucleic acid in the sampe and not just the RNAs... These are probably the three most common ways of measuring RNA and I guess as long as you use the bioanalyzer/gel to see the product quality (degradation) and have an idea of the conc using the spec or Qubit, you can still perform experiments well.

Dear all,

I just extracted the RNA from cancer cell line. I took them from my country and kept them in Trizol at room temperature for 2 days. Two day ago I extacted them. Unfortunately, all of them were degraded. I would like to ask someone who have the experience in RNA field, Trizol can not protect the RNA for a long time (two days) like my case? Because before I went to abroad to do my experiment, I asked someone in this lab and he told me RNA will be protected with Trizol. Anybody help me please.

hey guys,

thanks for the advice. i dilute my RNA and read my samples again. the purity amazingly increase!!!
i finally know the cause of my poor RNA purity. there is nothing to do with my technique while extracting RNA. it is just because of the dilution not enough.