5 replies to this topic

[thegene]

Hi mates.
I have performed transfection in HEK 293 cell with lipofectamine 2000 in 24-well format and i have performed RNA extraction using trizol.
What i have done is , I de-attached the cells using pippet tips then, centrifuged the cell+media and added trizol to the cells pellet.
The amount of trizol i added is related to 200000 cells, according to the manual, and that was 250 ul.
The problem is,  the upper phase that contains the RNA was small and hard to collect with out taking some of the organic phase.
My question is, if I increased the amount of trizol to double, do that affect the process in any way?

Best regards

[cotchy]

thegene, on May 20 2009, 10:09 AM, said:

Hi mates.
I have performed transfection in HEK 293 cell with lipofectamine 2000 in 24-well format and i have performed RNA extraction using trizol.
What i have done is , I de-attached the cells using pippet tips then, centrifuged the cell+media and added trizol to the cells pellet.
The amount of trizol i added is related to 200000 cells, according to the manual, and that was 250 ul.
The problem is,  the upper phase that contains the RNA was small and hard to collect with out taking some of the organic phase.
My question is, if I increased the amount of trizol to double, do that affect the process in any way?

Best regards

Hi thegene, why do you detach the cells first why not just add trizol to your dish after washing the cells with PBS?


If i remember correctly, it is recommended by invitrogen to use 1 ml of trizol per 3.5 cm  of surface area (a 6 well plates well area) so would be around 600 ul per well on a 24 well plate format, so are you adding enough of the trizol?

My suggestion would be to increase the amount of trizol/chloroform and see what affect this has on the aqueos layer.

However, if you remove the trizol/lystae to a single eppendorf for every well on the  24 well plate well then the aqueous layer is probably always going to be small because of the small volumes being used.

Cotchy

Edited by cotchy, 20 May 2009 - 03:13 AM.

[cotchy]

Further to my last email

Maybe you should perfom the transfections in a larger flask or dish

This way you will be able to add a larger volume of trizol and hence get a greater aqueous layer making it easier to remove without disturbing the fat/lipid layer.

Cotchy

[thegene]

cotchy, on May 20 2009, 03:31 AM, said:

Further to my last email

Maybe you should perfom the transfections in a larger flask or dish

This way you will be able to add a larger volume of trizol and hence get a greater aqueous layer making it easier to remove without disturbing the fat/lipid layer.

Cotchy

Thank you for the reply.
The thing is that invitrogen recommend this amount of trizol depending on the well size not on the cells number.
So that's why i am de-attaching the cells and transfer them to another tube, to be able to add more trizol, but i am afraid that trizol quantity affects the cells.

I am using 24 well format because i have a lot of transfections and it well be harder to do it in smaller formats.

Regards

[Bomber]

Hi,

as you already said, the amount of trizol is based on the surface area (10 cm2 = 1ml trizol).
This is what I would stick to in the first place.
Use respective amount of trizol for 24-well plate and let it stand 5 min at room temperature. After this you can either store the sample at -80°C and extract later or move on with extraction following the protocol.
Never had a problem with this from cell lines including HEK cells so far.
Another suggestion would be - if you have to collect RNA after different time points from the same plate:
the cells in the well next to the well filled with trizol will probably look very shity the next day, so either wahs this well very well with PBS (2x) or plate the cells in wells well seperated.

Good luck

[lsek]

Hi

U can add more vol of Trizol without detrimental effect on the RNA isolation. You won't lose the nucleic acid. More is better than less though.