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TRIZol Reagent additional reagents & protocol clarifications - (Jun/03/2011 )

Hi everyone,

I am very attracted in using TRIZol Reagent for RNA extraction from fish head kidney, spleen and thymus. Tthe kit available in my lab is Epicentre's MasterPure™ Complete RNA Purification Kit, the yield is very much lower compared to TRIZol (approximately 15ug for 5mg of sample).

I have not used both kit and I would certainly welcome opinions from experienced users for comparisons.

Questions:

1. The chloroform required in TRIZol protocol is it 100%?

2. Is it better to normal tips first for isolation or is highly recommended to use filtered tips right from the begining?

3. How long would it usually take for total RNA extraction plus RNA purification through Ambion's Turbo DNA-Free kit?

4. What is the quality difference between fresh sample and -80 snap frozen sample? Is it significant?

5. Is fine to homogenize the tissue + TRIZol Reagent with a vortex machine (ALERT: potential dumb question) or do I have to use the recommended homogenizers such as glass- 2 Teflon® or power homogenize?

-Jevandrix-

Jevandrix on Sat Jun 4 05:56:33 2011 said:


Hi everyone,

I am very attracted in using TRIZol Reagent for RNA extraction from fish head kidney, spleen and thymus. Tthe kit available in my lab is Epicentre's MasterPure™ Complete RNA Purification Kit, the yield is very much lower compared to TRIZol (approximately 15ug for 5mg of sample).

I have not used both kit and I would certainly welcome opinions from experienced users for comparisons.

Questions:

1. The chloroform required in TRIZol protocol is it 100%?

2. Is it better to normal tips first for isolation or is highly recommended to use filtered tips right from the begining?

3. How long would it usually take for total RNA extraction plus RNA purification through Ambion's Turbo DNA-Free kit?

4. What is the quality difference between fresh sample and -80 snap frozen sample? Is it significant?

5. Is fine to homogenize the tissue + TRIZol Reagent with a vortex machine (ALERT: potential dumb question) or do I have to use the recommended homogenizers such as glass- 2 Teflon® or power homogenize?


Hi,

I have used Trizol a lot and never had any real issues with it. In answer to your questions:

1. 100% chloroform
2. I always use regular DEPC treated tips for extraction. I use filter tips for the RT and PCR. Of course if you can aafford to then using filter tips for everything isn't a bad idea!
3. Can't help you with that one I'm afraid.
4. I've only used Trizol on snap frozen tissue and fresh cells so I have no direct comparison there, my frozen tissue always worked fine though.
5. I have always used a handheld glass homogeniser, I don't think a vortex would break down the tissue sufficiently.

Hope that helps!

-Hellie-

Hellie on Mon Jun 6 14:08:28 2011 said:


Jevandrix on Sat Jun 4 05:56:33 2011 said:


Hi everyone,

I am very attracted in using TRIZol Reagent for RNA extraction from fish head kidney, spleen and thymus. Tthe kit available in my lab is Epicentre's MasterPure™ Complete RNA Purification Kit, the yield is very much lower compared to TRIZol (approximately 15ug for 5mg of sample).

I have not used both kit and I would certainly welcome opinions from experienced users for comparisons.

Questions:

1. The chloroform required in TRIZol protocol is it 100%?

2. Is it better to normal tips first for isolation or is highly recommended to use filtered tips right from the begining?

3. How long would it usually take for total RNA extraction plus RNA purification through Ambion's Turbo DNA-Free kit?

4. What is the quality difference between fresh sample and -80 snap frozen sample? Is it significant?

5. Is fine to homogenize the tissue + TRIZol Reagent with a vortex machine (ALERT: potential dumb question) or do I have to use the recommended homogenizers such as glass- 2 Teflon® or power homogenize?


Hi,

I have used Trizol a lot and never had any real issues with it. In answer to your questions:

1. 100% chloroform
2. I always use regular DEPC treated tips for extraction. I use filter tips for the RT and PCR. Of course if you can aafford to then using filter tips for everything isn't a bad idea!
3. Can't help you with that one I'm afraid.
4. I've only used Trizol on snap frozen tissue and fresh cells so I have no direct comparison there, my frozen tissue always worked fine though.
5. I have always used a handheld glass homogeniser, I don't think a vortex would break down the tissue sufficiently.

Hope that helps!


Dear Hellie,

Thank you. You've been very helpful. However there is few more dumb questions (since I am new to RNA extraction):

1. Do we have to treat the tips with DEPC ourselves or is it sold in treated condition? If you have to do it, how would you treat tips with DEPC?

2. Can you give a rough estimation DEPC treated water, price? (I'm yet to get a quotation)

3. Have you ever worked with RNaseZap? If so what is the difference with DEPC treated water?

-Jevandrix-



Dear Hellie,

Thank you. You've been very helpful. However there is few more dumb questions (since I am new to RNA extraction):

1. Do we have to treat the tips with DEPC ourselves or is it sold in treated condition? If you have to do it, how would you treat tips with DEPC?

2. Can you give a rough estimation DEPC treated water, price? (I'm yet to get a quotation)

3. Have you ever worked with RNaseZap? If so what is the difference with DEPC treated water?


Hello,

Sorry had a busy couple of days and only just checked back! In response to your questions,

1. I am sure you can buy tips, it depends how much money you have! The method I use is, make up DEPC water, 1ml DEPC/litre dH20 and totally submerge the tips (and any eppendorfs etc you will be using) and then soak them overnight. You need to do this in a fumehood and it's a good idea to double glove, DEPC is pretty nasty. Once they've soaked the tips etc need to be autoclaved to break down the DEPC and they are then ready to use. The water should also be autoclaved before disposal or use (I used to use it for rinsing my homogeniser and any other kit I was using on the tissue).

2. Pricewise, DEPC is pretty cheap but offhand I don't know how much. I use bought in ultrapure DEPC H2O for putting in any actual reactions, ie dissolving RNA, making mastermix etc and that does cost a wee bit.

3. I have never heard of RNasezap but have used RNAse away which I am guessing is a similar thing, the reason I have never used that for tips etc is the cost. I use it to clean down the bench area prior to starting RNA work and to clean homogenisers etc between samples (then rinse with DEPC H2O before use).

I hope this helps, I don't know if this is the best way but it's always worked for me. Obviously if money is no object I would say buy in tips and water that are pre-treated and save yourself a lot of time and hassle but I've never had that luxury!

Hellie

-Hellie-

Hi,

you don't need filter tips at the beginning of the isolation (for putting trizol on the sample, transfer the whole to an eppi and so on...). they don't even need to be RNase free because the cells that you are breaking up are full of RNase so even if you have spurious amounts of RNase on your tips it is negligible.

I change to filter tips when i put on the chloroform (100%, of course), in fact it should be sufficient to use RNase free filter tips from the moment you are removing the aequous phase from the trizol.

And forget about treating tips or water with DEPC.... just use your normal tips at the beginning and then change to filtered ones (they should be anyway DNase/RNase free). Also use DNase/RNase free fabricated eppis right out of the bag (without autoclaving!).
Suitable water for RNA isolation is for example from Invitrogen Cat.No. 10977035 it is also quite cheap, 12.6 EUR for 500ml.

I'm doing RNA isolation since ~7 years using trizol, currently I'm using a similar reagent, its just ~3 times cheaper than invitrogen's trizol (peqlab trifast Cat.No. 30-2020).

-tea-test-