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RNA isolation from monocytes - (Aug/06/2009 )

Hi,
I try to isolate RNA from monocytes (700.000 cells) that are in culture for 2 hours. I tried different methods including Trizol, Qiagen RNeasy Mini kit and one kit from Ambion. We want to use the Qiagen kit for further experiments because it worked best.
My problems are the following with Qiagen:
I do the isolation according to the Qiagen protocol and I work highly sterile under a flow. I use the Qiagen shredders for homogenization, add ME to the RLT buffer and I wash twice with the RPE buffer. For the elution volume I used 20 - 50 ÁL which I leave for around 5 min and reuse after centrifugation for another elution step.
Nanodrop measurements show that my 260/280 ratio is often high (>1.9) but it varies a lot between different samples. My 260/230 ratio is extremely bad (<1.0). And my RNA yield is around 10 ng/ÁL independently of the elution volume.
Does anybody have any experience with RNA isolation from monocytes? Any suggestions, what I can do better?

Thanks a lot.

-Dr. Sue-

Dr. Sue on Aug 6 2009, 07:15 AM said:

Hi,
I try to isolate RNA from monocytes (700.000 cells) that are in culture for 2 hours. I tried different methods including Trizol, Qiagen RNeasy Mini kit and one kit from Ambion. We want to use the Qiagen kit for further experiments because it worked best.
My problems are the following with Qiagen:
I do the isolation according to the Qiagen protocol and I work highly sterile under a flow. I use the Qiagen shredders for homogenization, add ME to the RLT buffer and I wash twice with the RPE buffer. For the elution volume I used 20 - 50 ÁL which I leave for around 5 min and reuse after centrifugation for another elution step.
Nanodrop measurements show that my 260/280 ratio is often high (>1.9) but it varies a lot between different samples. My 260/230 ratio is extremely bad (<1.0). And my RNA yield is around 10 ng/ÁL independently of the elution volume.
Does anybody have any experience with RNA isolation from monocytes? Any suggestions, what I can do better?

Thanks a lot.




I don't have direct experience, but another person in my lab is trying to isolate mRNA from macrophages/monocytes. I know his 260/280s were pretty good, but not sure about his 260/230s. I think he got about 18 ng/ul of total RNA from 10^5 to 10^6 cells. He is doing it from mac/monocytes culture in poly-d-lysine plates. He was able to get pretty good efficiencies on a couple of trial qPCR runs over 6 or 8 logs of dilution on 18s and B-actin.

Now that I think about it a little more, I think he called Qiagen about some pretty poor 260/230 values, and was told by a tech that it was salts, but would not affect RT-PCR or subsequent real-time PCR, if that's what you plan on using the RNA for.

One thing you might try to clean it up a little more if you so choose is to use the RNA cleanup protocol for the RNeasy kit. I think it's towards the back of the manual. Basically, you just run the sample through a new column with a couple washes. I've had to do it before when my 260/280s were kinda low, and it improved them very well without much loss of yield.

-fishdoc-