Hi Pooja,

Your tissue (not RNA) is lost when you throw it in the TRIzol. 

I routinely isolate tissue from 5 day old zebrafish which have a weight of

Hi Pooja,

Your tissue (not RNA) is lost when you throw it in the TRIzol. 

I routinely isolate tissue from 5 day old zebrafish which have a weight of

Hi Bart,

I have stored tissue in RNA later (ambion) not in Trizol.  If you can share the protocol of rna isolation it will be really helpful.

Thanks,

Pooja

no problem

My colleague routinely uses Trizol for single snail neurons (they are huge, for neurons, but still very small). I don't know the exact weight of a single snail neuron, but a ganglion containing

Thanks to Bart and Ekaterina. I will try Trizol method on my tissue.

Depending of your downstream application, glycogen and t-RNA can be used as a carrier if your sample has low RNA content. However, linear polyacrylamide is the most inert carrier and recommended for critical applications.

Most common mistake with Trizol is to have too little Trizol per amount of tissue, which will often lead to A260/A80 ratios below 1.6. So having a bit too much Trizon does not hurt especially if you use polyacrylamide carrier. We used to make these ourselves but these are available commercially for this purpose, even in pink which makes life easier if you have a small pellet to recover. 

Hi!

I used to isolate RNA with trizol many times. I think 8-10 mg is enough.

Homogenize the tissue in liquid nitrogen in a crushing mortar (attention, the tissue bounces in it!), then wash the homogenized tissue from the morter with 1ml trizol into an 1,5 ml eppendorf tube. (The mortar should be steril and RNAse free!)

My protocol:

-stand 5-10 min at room tempreture

-add 200 ul cloroform, and shake it strongly for 15 sec

- 2-5 min stand at room temperature

-centrifuge: 12000 g, 15 min, 4 C

- the upper clean phase contains RNA, pipette it to a clean eppendorf tube

- add 500 ul isopropanol, suspend, 10 min stand at RT

- centrifuge 12000 g, 10 min, 4 C

- discard supernatant, dry the tubes edge

-1ml 75 % EtOH, suspend

- centrifuge 7500 g 5 min 4 C

- discard supernatant, dry the tubes edge

-dry the sample from EtOH : standing with tubes lid open, it i spossible to heat it to 30-37 Celsius (if you have vacuum dryer, than 10 min in it). attention, nothing should fall into the tube!)

-dissolve the RNA in 50 ul RNAse free water.

I hope I could help :)

M.

Hi, i work with trizol all the time and 2 mg tissue of prefrontal cortex, you shouldnt have any problem but use a good amount lile 750 ul to get enough aqueus phase and resuspend in 15 or 20 ul of depc water

Thank you Betina and Magdolna. Today, i isolated RNA from 6mg tissue from Bart's protocol I got decent amount of Rna. Thanks to Bart again.