Hey All,
So the goal of my latest set of experiments is to successfully extract great quality RNA from 1x10^6 cells (monocytes (THP-1) and macrophages(BAL washing's) using TRIzol Reagent for downstream qPCR.
I've struggled to get a concentration greater than 180ng/ul RNA eluted in 25ul.
Worse, more often than not I'm observing concentrations in the range of 60-70 ng/ul in 25ul which decreases a lot after DNase treatment (1/3 sometimes).
The protocol I have been using is as follows;
1) Remove media, wash cells and add 500ul TRIzol
2) Incubate for 5min at RT on 24 well plate, and then remove to new 1.5ml epi. I generally store down at -80C at this point and thaw on ice later.
3) After thawing sample, I then add 100ul fresh chloroform and shake hard by hand for 12-15 secs and incubate at RT for 3min
4) Spin 12000g 15min at 4C
5) Remove +-220ul aqueous phase to a new tube
6) Add 250ul 100% 2-propanol (generally use it at -20C)
7) Incubate samples for +-20 hours at -20C
8) Spin 12000g 20min at 4C, and discard supernatant
9) Add 500ul of 75% ethanol (RT), flick tube washing the sides
10) Spin 7500g 5min at 4C, discard supernatant
11) **Second ethanol wash (100%) - to improve A260/A280 ratio
12) Repeat step 10
13) Carefully remove supernatant
14) Air dry for 10min, or till pellet is dry
15) Elute in 25ul DEPC H20
16) Nanodrop
17)DNase Treat and nanodrop once more.
18) RNA stored at -80C
All reagents are made up fresh, or in DEPC water. Generally A260/A280 ratios range from 1.80-2.05.
Does the temperature of the reagents (other than TRIzol) matter?
Should I be adding any other steps which have helped you?
It seems a longer 2-propanol precipitation step at -20C is common (rather than the 10min precipitation at RT), but does this really make a difference?
In terms of disposables, I use new RNase free filtered tips and I've been wiping down all surfaces and pipettes with 10% bleach (NaOCl).
Thanks in advance!
After adding TRIzol to the plate of cell samples, be sure to agitate the plate for at least 30 seconds - to accomplish disruption/homogenization/agitation/lysis. Also, for the spins during the 75% and 100% ethanol washes, increase the g force up to 16,300 X g (instead of 7800 x g), this helps to slam the pellet against the walls/bottom of the tubes better. Generally 2.5 to 5 million cells would be more ideal for your parameters. And you are doing things correctly - the -20C 2-propanol is a good thing - and the 10 min incubation at room temp in 2-propanol can be done at -20C as well. Important: when you blank the Nanodrop for your post-DNase readings, be sure to blank it with the DNase treatment buffer etc. (e.g. everything that the samples are in, minus the sample, is what to blank the NanoDrop with; or else your readings could be thrown way off). Other than that, to me it sounds you are performing things correctly.
Your particular cells types may only be yielding 2 to 3 pg total RNA/cell as well...
Thank you for your suggestions Jack, I'll give them a try.
Arthur, I have run my RNA on our Agilent BioAnalyzer and I have been achieving RIN values ranging from 8.6-9.5 after DNase treatment.
Looking at the attachment below it just seems as though I'm losing RNA at some point, although I'm not sure where.
I'd be interested to see what your 230/260 ratios are, since in my experience cold isopropanol brings down an awful lot of guanidium salts (which TRIzol contains). I always use isoprop at room temperature, where it successfully precipitates RNA without salt co-precipitation, and reserve ice-cold/-20/-80 conditions for ethanol precipitations.
I also wouldn't bother DNAse treating, frankly, though a lot depends on your downstream plans for this RNA.
I've never had a problem with gunidinium thiocyanate salt precipitating out using -20C isopropanol. My total RNA always gives 260/280 readings of 2 to 2.12 and 260/230 readings of 1.85 to ~2. No problem with downstream qPCR (yet anyway). But, perhaps it is indeed best to do the 10 minute incubation in isopropanol at room temp (as is actually suggested in the TRIzol literature) now due to what John has brought up - better to be safe than sorry...
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