[Arabidopsis] RNA From Siliques

Ben Holt via arab-gen%40net.bio.net (by benholt from ou.edu)
Mon Jun 4 08:46:56 EST 2007


Apparently the attachment I sent regarding collecting RNA from =20
siliques was not accepted by the listserv (it was attached, but =20
apparently they get removed?). Anyway, several people have already =20
asked for the protocol, so I have just pasted it as text below. And =20
don't thank me for the protocol - I am just passing on something I =20
saved in 2003! Thanks to Dr. Tamara Western.


Good Luck,
Ben Holt


RNA Isolation (Acid Phenol:LiCl) =96 Tamara Western

1.  Grind tissue into powder in liquid N2 in small mortar & pestle =20
with the addition of a small amount of sand to help grinding.

2.  Pre-cool tubes & scoopula by sitting in N2. Transfer dry powder =20
to tubes & leave to sit open in N2  until finish all tubes. Once all =20
samples ready, take each into phenol (Step 4) separately & at that =20
point can sit on ice while do next (speed of getting into phenol =20
important).

3.  Add 1 ml REB, vortex quickly to mix. NB good idea to give samples =20=

a chance to =93breathe=94 on ice before add REB because if any N2 left =20=

will cause REB to bubble out of tube (30 sec or so).

4.  Add 1 ml 2:1 phenol:CIA. Vortex quickly & thoroughly (1 min). =20
Store tubes on ice until other samples ready, then re-vortex all 1 min.

5.  Spin 15 min at 4oC .

6.  Remove supernatant to fresh tube (~1 ml). Extract with 1 vol 1:1 =20
phenol CIA. Vortex 1 min. Spin 10 min at 4oC .

7.  Remove supernatant to fresh tube (~950 ml). Extract with 1 vol =20
CIA. Vortex 1 min. Spin 8 min at 4oC.

8.  Remove supernatant to fresh tube (~850 ml). Add 10M LiCl (DEPC) =20
to supernatant to reach 2M LiCl (=93drop by drop while gently vortexing =20=

to prevent massive co-precipitation of DNA=94 =96 wrt vortexing, used =20=

shake #3). Rule of thumb to get 2M LiCl: take 2x final volume & =20
divide by 10 to get 10M LiCl to use, need to do by iteration since =20
final volume changes with LiCl added. Starting point: ~200 ml 10M/1 =20
ml final volume. Thus have determined: if 850 ml spnt + 210 ml 10M =3D =20=

1060 ml final =3D ~2.0M final conc.

9.  Precipitate on ice 2 h. NB =96 inverted ~5x first to make sure =20
completely mixed.

10.  Spin 10 min. Should see white pellet. Wash pellet with 1 ml 70% =20
ethanol (cold, in DEPC water). Air dry 5-10 min.

11.  Dissolve pellet in 200 ml DEPC-water.

12.  Add 20 ml 3M NaOAc pH 5.2 (DEPC) & 500 ml cold 100% ethanol. =20
Mix. Let sit on ice 20 min.

13.  Spin down 20 min at 4oC.

14.  Optional step to remove excess polysaccharides (highly =20
recommended to do at least 1x for siliques). Add 330 ml 3M NaOAc pH =20
5.2 (DEPC) to pellet. Vortex vigourously to resuspend pellet (break =20
it up). Let sit 10 min at room temp. Spin 10 min at 4oC. Remove =20
supernatant. (Should see decrease in pellet size. If need very pure =20
silique RNA, repeat before continuing to 70% ethanol wash.)

15.  Wash with 1 ml 70% ethanol (cold, in DEPC water). Air dry 5-10 =20
min. NB used aspirator to remove final drops of ethanol, only needs =20
~5 min dry.

16.  Dissolve in 50-100 ml DEPC-water. If need very pure RNA, perform =20=

secondary purification through Qiagen RNeasy columns using the Clean-=20
up protocol (NB: divide the prep onto 2 columns, incubate elution =20
water on membrane 10 min before spinning down).
Solutions


REB:             25 mM Tris-HCl pH 8.0
             25 mM EDTA
             75 mM NaCl
             1% SDS
Acid Phenol =96 Sigma P4682 (Phenol equilibriated with NaCitrate pH 4.3)
CIA: 24:1 chloroform:isoamyl alcohol
10M LiCl (DEPC-treated)
3M NaOAc pH 5.2 (DEPC-treated)
DEPC-water


Notes:

             Modified from a protocol from Jerome Giraudat. In my =20
original trial, I used approx 100 mg of inflorescences + siliques at =20
~7 days after pollination (dap) & got ~1mg/mg tissue for =20
inflorescences & 0.4 mg/mg for siliques. In later trials I've found =20
that it works best for 100-200 mg siliques (probably due to tissue =20
loss in transfer from mortar & pestle). A typical yield is 50-80 mg =20
from ~120-150 mg siliques (approx 20-30 at 7 dap). The A260/280 is =20
typically 1.96-2.01, the RNA looks good on a formaldehyde gel & has =20
been successfully used for both one-step & two-step RT-PCR. I have =20
successfully used this for siliques up to 10 dap, at which point all =20
the mucilage is present and the secondary cell walls of the epidermal =20=

& palisade layers of the seed coat are in mid to late synthesis =20
(embryo ~ upturned U).



=3D=3D=3D=3D=3D=3D=3D=3D
Ben Holt
Assistant Professor
University of Oklahoma
Department of Botany and Microbiology
GLCH Rm 219
770 Van Vleet Oval
Norman, OK  73019
Phone (405)325-9018
FAX   (405)325-7619
http://www.ou.edu/cas/botany-micro/faculty/holt.html






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