In general the use of guadinium/phenol based kits such as Triazol for Sigma
give better results than this, the original method. But it can be expensive
for large numbers of samples. So for what its worth, here is my original
methods with a slant toward fish RNA sources.
Fish tissue RNA purification
LIVER OR TISSUE: 50-100mg of liver provides plenty of RNA and is homogenised
in 4ml polypropylene tubes in 1ml (or 300-500mg in 4ml) of homogenisation
buffer for 10sec using the polytron at full speed for 10sec. Spin these
homogenates at 5000g for 5mins and decant the supernatant to fresh tubes.
Othe tissues such as muscle for example are much lower in RNA content,
tougher to homogenise and should be minced first.
CELL CULTURE: Similarly one small cell culture tube (25cm2)provides
enough RNA for 1-2 Northern blots. Rinse the cells in PBS then add 400µl
of guanidinium thiocyanate RNA homogenisation solution and shake for 5mins,
tipping the tube at least once to make sure that the gelatinous solution
has been passed over the whole plate. Place the flasks upright and tilted
to collect the extract and then pippette into microtubes. Pass this solution
twice through a narrow gauge needle to shear high molecular weight DNA.
FISH LARVAE: Marine fish larvae should have as much of the seawater removed
first. This can be achieved by first filtering out the larvae over nylon
mesh, adding a wet weighed portion to a polypropylene tube and topping
up with fresh water and immediately spinning the larvae down. The larval
pellet can now be taken up in RNA homogenisation buffer and homogenised
as described for liver.
Any of these homogenates may be stored at -20°C or even better at -70°C
for at least 2 months at this stage. Subsamples can then be taken from
these homogenates and further processed; eg for convenience RNA can be
purified from 500µl subsamples transferred to microtubes. Any of
these procedures may be scaled up for larger samples.
For every ml of homogenate, add 100µl of 2M sodium acetate, pH 4.0
and mix. Then add 800µl per ml of phenol (unbufferred, water saturated
eg Aquaphenol fron Appligene) and whirlimix vigorously. Add 200µl
per ml of chloroform and whirlimix vigorously. Spin at 5000g for 10 minutes
and transfer the aqueous phase to a fresh polypropylene tube. If the aqueous
phase is still brown, extract with an equal volume of 50% phenol, 50% chloroform.
Repeat until the upper phase is clear.
Add and equal volume of isopropanol (usually about 1.2ml per ml of original
homogenate buffer) and precipitate in the freezer for at least 1 hour.
Spin at 5000g for 15mins and pour off the supernatant. Drain the tubes
upside down on paper towel and wipe the inside walls with sterile tissue
(Kimwipes) being careful not to disturb the pellet. The pellets are whitish,
small and smeary and can be quite loose and slippy. Spinning the precipitations
harder (eg in the Sorvall) may improve this situation.
Optional step for purer samples. (not necessary for blotting or gels) Resuspend
the pellet in 200µl of RNA homogenisation buffer. Use only factory
sterile plasticware, MilliQ water and highest purity solvents from now
on. Transfer to an eppendorf (up to three tubes-ie three culture flasks
may be combined at this point) and an equal vol of isopropanol. Precipitate
the RNA in the freezer for 30min. Spin the tubes at full speed in a microfuge
for 10mins and carefully pippete off as much of the supernatant as possible
Wash the pellet by adding 1ml of ice cold of 70% ethanol for 2mins, respin
briefly and remove ethanol, dry on the bench for 10-15min, covering open
tubes with a sterile tissue, and resuspend in 5µl RNAase free water
per 25cm2 of culture or 1µl per mg of liver. ( other tissues and
fish larvae should be tested first to see whether the method works and
to get some idea of the yields). Measure the absorbance of 100 fold dilutions
at 260nm and 280nm (Donít do this for culture flaks as not enough material
is present. The ratio 260/280 gives a measure of purity (R = 2 is regarded
as pure, 1.8 is usually acceptable) and an A260 of 1 is equivalent to an
RNA concentration of 30µg/ml.
RNA homogenisation buffer is 4.5M guanidinium thiocyanate (or 6M guanidinium
hydrochloride), 25mM sodium citrate, pH 7.0, 0.5% sarkosyl stored at room
temp with DTT added to 10mM from frozen 1M stock immediately before use.
Alternatively use a commercial mix (eg Triazol or RNAzol) which are
essentially the same as described above but can give better results ( for
a lot more cost)
It is important to wear gloves at all stages, firstly to afford protection
from guanidinium thiocyanate and phenol and secondly to minimise RNAase
MilliQ (or eqivalent grade) water is OK to use directly if collected
in factory sterile plastic. DEPC can be used to treat solutions and plastics
or glass if required. DEPC treatment of solutions involves adding 0.1%
DEPC to the solution, shaking for 1 hour and autoclaving. Tips and vials
are shaken in suitable containers in a suitable volume of 0.1% DEPC/ water
for 1 hour, drained and autoclaved. Excess moisure may be removed by leaving
in the drying cabinet with loose lids for a while.
Repeated washes with 70% ethanol can often result in cleaner preps.
Attempting to homogenise too much tissue in too small volumes of buffer
will result in poor preps, RNA pellets resistant to resuspension, and low
purity ratios. A 10% (w/v) homogenate is about the upper limit for good
liver and larval RNA.
Drying RNA pellets too much will also make them resistant to resuspension
although traces of ethanol can give similar problems. Therefore it is recommended
that pellets are allowed to dry in air for about 15 mins or longer if necessary.
Dry means no ethanol left- pellets will still be moist with residual water.
It is critical that all samples are treated equally and great care is
taken to avoid RNAase contamination, therefore it is recommended to avoid
working up large numbers of samples at one time. It is better to get good
samples over a few days than many poor samples in a morning. It is also
important to test a previously unused source material to check that it
is possible to get good RNA before embarking on a full scale experiment.