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ASK FOR HELP: why I can not get total RNA from human liver - (Jun/23/2006 )

Hi Dear all, smile.gif smile.gif

I just moved to a small new lab, and we are trying to do a microarray by using the human tumor liver sample.

Yesterday I took 8 human liver tumor sample from -80C which were stablilized by RNAlater. Each sample is around 20mg. I ues Qiagen RNeasy mini kit. After adding 600ul RLT buffer, I used a mechanical homogenizor (Fisher Power Gen 125) to homogenize my sample. I according to the protocol to finish the total RNA preparation. I use 50ul nuclease free water to elute my RNA. After I used Agilent 2100 to detect the RNA quality and quantity of my RNA. Unfortunately, no of them is good. Their Con. are around 80-120ug/ul, and four of them have no 18srRNA and 28srRNA band, others have 18srRNA and other small RNA bands. I feel really frustrated about these results. sad.gif sad.gif

I carefully think my whole experiment and I think probably these problems are the reasons:

1. Homogenize time. I have 8 samples totally, and also spent half an hour to do other things after the homogenization, probably the homogenized sample stayed in ice too long before I continue the next step.
After I spinned the homogenized solution, I can saw there are around 30-40ul pellet. After I add the supernatant to column and spin, the column looks very dirtly. Probably the tissue are not homogenized enough.

2. I just use dH2O from Gibco to make 70% ethanol, and also for the homogenizor, I just use dH2O wash and Buffer RLT wash between each sample.

Can anybody give me some suggestion? I do need help for the homogenization of human liver sample. Someboday said mouse liver is more easy than human liver.

Thanks for any suggestion! rolleyes.gif rolleyes.gif

-BLUE-SNOW-

Hi Blue Snow-

I isolate RNA from human primary hepatocytes, and I also use the RNeasy Mini Kit..You should be able to recover LOTS of RNA from the liver.

I had problems for a long time with recovery, mostly likely due to the matrigel on the cell culture plates. But one thing that made all the difference for me was to add the Proteinase K digestion in with the standard RNeasy protocol (the method is in Appendix C in the Handbook). I am kinda just guessing here, but I wonder if the liver tumor cells are particularly fibrous? That may explain why your column looks dirty. In my experience, it isn't too hard to clog the column and lose your RNA.

Also, I agree that you should use nuclease-free water for pretty much everything.

Do you do the DNase digestion step? I have found my DNase to degrade my RNA in the past.

You may consider starting with less than 20 mg tissue: I wonder if you have too much RNA and that could also clog your columns? (I have recovered loads of RNA from liver tissue (not cultures) in the past, but I used Trizol.)

Finally, I don't think letting the samples sit on ice for 1/2 an hour in Buffer RLT will cause any harm. I have let RNA samples sit in Buffer RLT at room temp for about that long with no problems. Homogenization buffer is usually pretty protective.

Good luck!

-soluene-

Hi soluene,
Thank you so much for your nice reply.
Probably I should consider protainase K didestion at the same time.
You said you recovered loads of RNA from liver tissue in the past by Trizol. Do you remember that protocol? Do you think homogenization enough or not is very important? If over homogeniztion I am afraid my RNA will degrade. I am just not sure the degree of homogenization.

I personally think probably Trizol is more better than those Kit for RNA preparation.

Thanks again for your input! rolleyes.gif rolleyes.gif








QUOTE (soluene @ Jun 23 2006, 11:22 AM)
Hi Blue Snow-

I isolate RNA from human primary hepatocytes, and I also use the RNeasy Mini Kit..You should be able to recover LOTS of RNA from the liver.

I had problems for a long time with recovery, mostly likely due to the matrigel on the cell culture plates. But one thing that made all the difference for me was to add the Proteinase K digestion in with the standard RNeasy protocol (the method is in Appendix C in the Handbook). I am kinda just guessing here, but I wonder if the liver tumor cells are particularly fibrous? That may explain why your column looks dirty. In my experience, it isn't too hard to clog the column and lose your RNA.

Also, I agree that you should use nuclease-free water for pretty much everything.

Do you do the DNase digestion step? I have found my DNase to degrade my RNA in the past.

You may consider starting with less than 20 mg tissue: I wonder if you have too much RNA and that could also clog your columns? (I have recovered loads of RNA from liver tissue (not cultures) in the past, but I used Trizol.)

Finally, I don't think letting the samples sit on ice for 1/2 an hour in Buffer RLT will cause any harm. I have let RNA samples sit in Buffer RLT at room temp for about that long with no problems. Homogenization buffer is usually pretty protective.

Good luck!

-BLUE-SNOW-

Absolutely, I still use the Trizol protocol actually! I use a hybrid protocol that combines Trizol extraction and the RNeasy method.



Here is a link to a thread where the Trizol protocol is attached. I think homogenization is very important! I used a glass-teflon homogenizer (cleaned with RNase-Zap) when I was isolating RNA from tissues.



I haven't really tried anything but Trizol so I can't personally compare other methods; however my supervisor stuck like glue to this isolation method, even when I was trying to convince him to try other kits. Now I see why!

-soluene-

Thank you for providing the link! :lol laugh.gif
I am also considering combination Trizol extraction and RNeasy mini kit together. Do you think it is OK if I use Trizol extraction to get my RNA pellet and then use RNeasy column to purify the RNA?
I believe Trizol extraction more than the RNeasy kit.


QUOTE (soluene @ Jun 23 2006, 12:59 PM)
Absolutely, I still use the Trizol protocol actually! I use a hybrid protocol that combines Trizol extraction and the RNeasy method.



Here is a link to a thread where the Trizol protocol is attached. I think homogenization is very important! I used a glass-teflon homogenizer (cleaned with RNase-Zap) when I was isolating RNA from tissues.



I haven't really tried anything but Trizol so I can't personally compare other methods; however my supervisor stuck like glue to this isolation method, even when I was trying to convince him to try other kits. Now I see why!

-BLUE-SNOW-

Actually, that is exactly what I do. I get my RNA pellet from the Trizol extraction, wash it twice in 75% EtOH as per the Trizol protocol...but then instead of resuspending my pellet in RNase free water or 1X TE, I resuspend my pellet in 300 ┬ÁL Buffer RLT. In Buffer RLT, I do the PK digestion and then add that solution to an RNeasy column...and follow through with the RNeasy protocol.

You may also consider resuspending your pellet in 1X TE or RNase free water, and following through with the RNeasy MinElute Clean-up kit. This method does not work for me because it does not have a PK digestion, which I need to digest Matrigel. But I have cleaned up liver tissue RNA (no matrigel) with the MinElute method with pretty good results (but still about 20% loss).

-soluene-