RNA extraction from alginate beads - (Jul/16/2007 )
hi everybody,
I'm using trizol rna extraction for a long time without problems. since i'm trying to extract rna from cells embedded in alginate beads, the reverse transcription doesn't work, except i use only one tenth volume of rna for reverse transcription.
After dissolving of the alginate beads with dissolving buffer (55 mM sodium citrate, 30 mM EDTA, 0.15 M NaCl) cells are centrifugated and washed two times in PBS. Then the pellet is dissolved in 1 ml of tri reagent.
My first idea was that some remaining sodium citrate or EDTA could inhibit the reverse transcription, but the pellet is washed two times in 10 ml PBS, so it could hardly be the cause.
Does someone have an idea?
Thanks a lot
I'm using trizol rna extraction for a long time without problems. since i'm trying to extract rna from cells embedded in alginate beads, the reverse transcription doesn't work, except i use only one tenth volume of rna for reverse transcription.
After dissolving of the alginate beads with dissolving buffer (55 mM sodium citrate, 30 mM EDTA, 0.15 M NaCl) cells are centrifugated and washed two times in PBS. Then the pellet is dissolved in 1 ml of tri reagent.
My first idea was that some remaining sodium citrate or EDTA could inhibit the reverse transcription, but the pellet is washed two times in 10 ml PBS, so it could hardly be the cause.
Does someone have an idea?
Thanks a lot
How long did you culture the beads? Do you have the same problems at T=0?
I use the RNeasy (the normal rneasy mini kit) from qiagen. I just put the whole beads in the first lysis buffer (with beta-mercaptoethanol), it needs a bit of pipetting to dissolve the beads, but it works very well.
what's the volume of your pellet? i would recommend to use 1mltrizol per 50µl cell pellet.
2 washes at 10ml is ok to dilute at extreme remaining EDTA.
You may have RNA degradation? contaminants (how much is OD230)?
The culture time in alginate beads has no influence on the inhibitory effect.
I'm using 1ml trizol per 1x10^6 cells.
I have already tried to put the alginate beads directly in trizol, but the inhibitory effect was greater than first dissolving the beads and then washing the cells.
Furthermore I purified the isolated RNA with RNeasy kit from quiagen or performed a second trizol isolation. All with no or little success.
@aspergillie
What alginate do you use and how many beads do you use for isolation with the RNeasy kit? I'm using Alginic acid sodium salt from brown algae, low viscosity, plant cell culture tested, powder (Sigma).
Produced proteoglycans could give an inhibitory effect, but if the culture time has no influence, then this is not the case.
I use alginate: Keltone LVCR (Kelco, Pharmaceutical Ingredients) (1,2% in PS), I seed 2.5-4 x106 cells/ml alginate. I pass them through a 23G needle and I use about 20 beads for RNA isolation. I just add the lysis buffer and first dissolve them with a p1000 pipet and afterwards with a 23G needle (I use 600µl of lysis buffer). I never tried this with Trizol.
We also had some problems isolating RNA from whole tissue. We could not measure any RNA using nanodrop, but we could measure 18S and 28S by qReal-Time PCR and on picochips (to measure the quality of your RNA by an electropherogram). Maybe you could have some problems like this?