I tried to extract RNA at the beginning of my experiment (0h), and now the cell concentration is low (about 10exp5). Following the Trizol instructions I add 800µL instead of 1mL, but still have low RNA yields.
I'm thinking if I could use a carrier like glycogen to improve the RNA concentration, and maybe try with a commercial kit?
Any ideas on how to improve the RNA concentration?
With only 100,000 cells, RNA yields will be reasonably low anyway, so bear that in mind. Given that it's a time zero sample, there is no reason not to simply use more cells.
Also, 0.8-1ml of TRIzol is almost certainly overkill for cell numbers that low. I routinely use 100-200ul of TRIzol for 12- or 6-well plate RNA extractions, which has the added benefit of being cheaper on the TRIzol budget, too.
Glycogen will certainly help, though. (See below)
For a 12-well plate (assuming adherent cells) my standard approach is to remove media, add 100ul TRIzol (essentially: sufficient to cover the surface), detach/disrupt cells by agitation with a cell-scraper, and collect the TRIzol into a 1.5ml epi (I recommend 1.5ml epis rather than 2ml, as it makes phase-separation clearer).
Following the standard TRIzol protocol, add 20ul chloroform, vortex, spin, and collect aqueous phase (carefully).
Collecting the aqueous phase from these cleanly is non-trivial, as the volumes are small and likelihood of protein/phenol carryover is relatively high, thus I usually don't worry too much, and perform an additional cleanup step instead:
Collect the aqueous phase without too much concern over protein/phenol carryover, (you should be able to get around 50ul) and dispense into 450ul of DEPC H2O. This pads out the volume, which makes the subsequent steps easier.
To this add 500ul chloroform (so 1:1 ratio) and vortex vigorously, then spin as before (13krpm in a benchtop centrifuge, 4 degrees, 10mins). It should be relatively easy to then remove 450ul of the subsequent aqueous phase, which should be much, much cleaner. To this, add :
10ug of glycogen (usually supplied at 20mg.ml-1, so 0.5ul)
45ul of 3M sodium acetate pH 5.5 (because you've diluted your trizol prep, additional salt is necessary)
450ul of room temperature isopropanol
Mix well (vortex or shake) and incubate at room temperature for 10-20mins. Room temperature is important: isopropanol precipitates salt very easily at low temperatures.
Collect RNA at 13krpm, 4 degrees, 10mins, and wash the pellet (gently) with 70% ethanol, twice, before air drying for 15mins and dissolving in DEPC H2O.
I usually obtain 10-20ug of RNA this way, and thus find that dissolving the pellet in 20ul H2O gives a final [RNA] suitable for easy cDNA synthesis (0.5-1ug.ul-1). While this may vary somewhat depending on how transcriptionally and translationally active your cells are, your yields should be in this rough ballpark.
With only 100,000 cells, RNA yields will be reasonably low anyway, so bear that in mind. Given that it's a time zero sample, there is no reason not to simply use more cells.
Also, 0.8-1ml of TRIzol is almost certainly overkill for cell numbers that low. I routinely use 100-200ul of TRIzol for 12- or 6-well plate RNA extractions, which has the added benefit of being cheaper on the TRIzol budget, too.
Glycogen will certainly help, though. (See below)
For a 12-well plate (assuming adherent cells) my standard approach is to remove media, add 100ul TRIzol (essentially: sufficient to cover the surface), detach/disrupt cells by agitation with a cell-scraper, and collect the TRIzol into a 1.5ml epi (I recommend 1.5ml epis rather than 2ml, as it makes phase-separation clearer).
Following the standard TRIzol protocol, add 20ul chloroform, vortex, spin, and collect aqueous phase (carefully).
Collecting the aqueous phase from these cleanly is non-trivial, as the volumes are small and likelihood of protein/phenol carryover is relatively high, thus I usually don't worry too much, and perform an additional cleanup step instead:
Collect the aqueous phase without too much concern over protein/phenol carryover, (you should be able to get around 50ul) and dispense into 450ul of DEPC H2O. This pads out the volume, which makes the subsequent steps easier.
To this add 500ul chloroform (so 1:1 ratio) and vortex vigorously, then spin as before (13krpm in a benchtop centrifuge, 4 degrees, 10mins). It should be relatively easy to then remove 450ul of the subsequent aqueous phase, which should be much, much cleaner. To this, add :
10ug of glycogen (usually supplied at 20mg.ml-1, so 0.5ul)
45ul of 3M sodium acetate pH 5.5 (because you've diluted your trizol prep, additional salt is necessary)
450ul of room temperature isopropanol
Mix well (vortex or shake) and incubate at room temperature for 10-20mins. Room temperature is important: isopropanol precipitates salt very easily at low temperatures.
Collect RNA at 13krpm, 4 degrees, 10mins, and wash the pellet (gently) with 70% ethanol, twice, before air drying for 15mins and dissolving in DEPC H2O.
I usually obtain 10-20ug of RNA this way, and thus find that dissolving the pellet in 20ul H2O gives a final [RNA] suitable for easy cDNA synthesis (0.5-1ug.ul-1). While this may vary somewhat depending on how transcriptionally and translationally active your cells are, your yields should be in this rough ballpark.
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