Note: Use Rnase-free filter/barrier tips for every step of the following protocols!
Wear only nitrile (purple) gloves, as latex may cause fluorescent background.
RNA Extraction
Number of flies
Trizol
Chloroform
Isoproponal
1
50 μl
10 μl
25 μl
~5
100 μl
25 μl
62.5 μl
~10
200 μl
50 μl
125 μl
~20
500 μl
125 μl
250 μl
~40
1000 μl
250 μl
500 μl
1. Add 50 μl Trizol to each tube and grind fly completely
2. Incubate at room temp. for 5 min.
3. Centrifuge 12,000 g at 4º C for 10 min., transfer supernatant to a clean tube.
4. Add 10 μl chloroform (be careful it drips from tips), mix well by shaking the
tubes vigorously for 15 sec by hand, incubate at room temp. for 3 min.
5. Centrifuge 12,000 g at 4º C for 10 min, transfer the aqueous (upper) phase to a
clean tube (must be Rnase-free at this stage!). Be careful not to transfer any of
the interphase.
6. Add 25 μl isopropanol (invert tubes twice), incubate at room temp. for
exactly 10 min.
7. Centrifuge 12,000 g at 4º C for 10 min, remove supernatant (a clearly-visible
white pellet should remain – not so visible if <5 flies used).
8. Wash the pellet with 100 μl 70% ethanol prepared with Rnase-free water
(which is stored in -20 º C). Vary amount according to number of samples- at
least same volume as trizol.
9. Gently molest the pellet so that it is broken up.
10. Centrifuge 12,000 g at 4º C for 10 min.
11. Wash the pellet at least twice.
12. At this stage, the sample is stable in ethanol at -80º C. Store the sample until
shortly before beginning with cDNA synthesis.
13. Remove the ethanol completely and air dry for ~5-10 min. It may be helpful
to first remove the ethanol, then briefly centrifuge and again remove any
ethanol that accumulated from the spin. Do not over-dry or samples may be
difficult to resuspend (better to have a tiny bit of ethanol left than over dry).
14. Resuspend the pellet in ~5 μl of RNA storage solution. RNA storage solution
is more stable than water for long term storage. Dissolving may be aided by
several tapping + brief centrifugation’s and/or heating at 37-55º C until
dissolved. Can be difficult to re-suspend. Be patient! (1 fly in 5 μl, ~5 flies in
10 μl, 10 flies in 15 μl, >15 in full 30 μl).
15. Should be left with ~5 μl of sample at a concentration of 1-5 ug/uL
Small volume RNA extractions can then be stored in (RNase free) strip tubes in -80º
C freezer.
1 μl of extraction can be used in RT. RT (25 μl) can then be diluted to between 1 in
5/1 in 10 for PCR.
If desired you can quantify RNA using nanodrop.
-260/280 (RNA quality) ideal ~1.9-2.1.
-260/230 (protein contamination) >1.8, tend to be less (~1.3) but is fine.
You can also run 1 uL on a gel, rRNA band to check for degradation.
Equipment
TRI Reagent 100ml
Isoproponal (2-proponal)
Chloroform (CHCl3)
Micro test tube 3810X
Tube pellet pestle 0.65mm
THE RNA Storage Solution
Applied Biosystems UK
sigma
sigma
Eppendorfs
Anachem.
Applied Biosystems UK
AM9738
I9516
C2432
DIS-800-080L
1004-32
AM7001