5 replies to this topic

[kbolt]

I am wanting to get the best quality and quantity of RNA possible.  I have a Trizol protocol that states to use about 10 ul Trizol / 1 mg of tissue, and I am wondering if this holds true for brain tissue or if this is a rule of thumb for peripheral tissue.  The tissue sample I have is smaller than 1 mg (it's just a punch of the rat brain).  A girl in my lab has extracted RNA from the same type of brain tissue and used 250 ul Trizol for the same small amount of tissue (obviously disreguarding the 10ul/1mg).   She successfully extracted RNA but it wasn't very high quality and quantity.  I am sure there are many reasons for her results, but I am just brainstorming the optimal Trizol amount currently.  So my question is...how much Trizol should I use for rat brain tissue?  If anyone can give me a little guidance I would appreciate it!

[suhasbn]

just stick to the manufacturers protocol (trizol) it shud work fine. good luk

[badcell]

With Trizol, I believe that the more quantity of the reagent you use, the better the results you get. I work with small tissue samples and always use 1 ml Trizol. Don't forget you need to add a carrier such as glycogen in the isopropyl precipitation step.

[kbolt]

badcell, on Jan 21 2005, 11:35 AM, said:

With Trizol, I believe that the more quantity of the reagent you use, the better the results you get. I work with small tissue samples and always use 1 ml Trizol. Don't forget you need to add a carrier such as glycogen in the isopropyl precipitation step.

Thanks for the help!  One more thing...glycogen isn't in my protocol.  How much glycogen should I use in the isopropyl precipitation step...and what does it do exactly?

[pcrman]

Glycogen is a carrier for the precipitation of nucleic acids (DNA or RNA).  It is not a must for RNA extraction with trizol. I've never used trizol but used a similar reagent called TriReagent. I found it unnecessary using glycogen with Trireagent.

You can use 20 ug per sample. That amount will allow you to precipitate pg-amount of RNA from a volume of 1 ml.

[badcell]

Quote

Glycogen is a carrier for the precipitation of nucleic acids (DNA or RNA). It is not a must for RNA extraction with trizol.

Yeah, you don't need to add glycogen if your sample is big enough, but I found that glycogen is necessary when working with small samples of tissue or cells. It increases a lot the quantity of RNA you can extract. I usually work with as few as 15000 cells and I got enough RNA to measure OD, run in an gel and do RT. I would certainly add glycogen for a sample of 1 mg tissue.