4.4@ RNA Extraction
A.
Precautions
(1) Use
gloves, disposable tubes (usually not necessary to autoclave, use RNase-free
tubes)
(2) Make a
distinction between RNA utensils and reagents and those used for other purposes
(Do not use RNA utensils for other purposes. It is better to make special shelf
for RNA reagents)
(3) Do not
open the caps of the bottles of RNase and Pronase in the laboratory.
(4) Do not
weigh RNase, DNase, and Pronase.@
Believe the label and dissolve the contents at a time with appropriate
buffer.
(5) Do not
discard high concentrations of the solution or used tubes in the lab.
(6) Be
careful of contamination. Use species-specific mortars and pestles if
necessary.
(7) Plant
tissue is the biggest source of RNase, so mix tissue with buffer as quickly as
possible.
(8) You
yourself are another source of RNase. Keep your mouth shut!!
B.
Pretreatment
(1)
Sterilize all utensils by autoclaving (120˚C 20 min) or oven (200˚C 3
hours). Plastic tubes do not require pretreatment.
(2)
Inactivate RNase with DEP
(Diethylpyrocarbonate):
All
reagents except Tris
@@@@@@@@@@@@@@@ Add 2%
(v/v) DEP in Draft
@@@@@@@@@@@@@@@ Keep at
least 2 hours at room temp in Draft
@@@@@@@@@@@@@@@ Autoclave
(121C 40min)
For Tris
Use RNase free reagents (usually there is no problem to use
regular grade Tris, but do not use the bottle for other purposes)
@@@@@@@@@@@@@@@ Dissolve
in DEP treated H2O with sterilized utensils
@@@@@@@@@@@@@@@ Adjust
pH with pH paper or clean pH electrode
1jRNeasy
(QIAGEN) --- for protonemata: for RACE, RT-PCR
2jISOGEN,
ISOGEN-LS (NIPPON GENE, Tokyo, JAPAN) --- for protonemata, sporophytes: for
RACE, RT-PCR
3jGuSCN
and CTAB method --- for protonemata: for northern, RT-PCR
4jDYNABEADS
mRNA DIRECT kit (DYNAL) --- for both protonemata and gametophores: for any
purposes
You
can extract relatively pure total RNA from protonemal tissue, while it may be
rather difficult to get pure RNA from gametophores. During RNA isolation steps
from gametophores, a polysaccharide-like gelatinous contaminant co-precipitates
with extracted RNA after centrifugation, and this pellet never dissolves.
Centrifugation is not involved in method 4), and is useful to extract RNA from
gametophores. Total RNA from young sporophytes with archegonia still attached
was successfully extracted by methods 1) and 2).
We
use different RNA extraction methods depending on purpose.
For RACE
Total
RNA and mRNA extracted by 1) and 3) work for the 3'RACE System kit (Invitrogen)
and Marathon cDNA amplification system kit (CLONTECH). Total RNA by 3) may
sometimes give trouble because of purity.
@
For Northern hybridization
It
is better to use about 1 µg poly(A)+RNA per lane for northern blotting,
although 10 µg of total RNA per lane may work.
DNase
treatment is necessary for every method.
1)@ RNeasy (QIAGEN)
Follow
manufacturer's manual.
Note:
-
This method is as easy as method 2).
-
Avoid overloading sample onto column.
- genomic DNA usually contaminates the RNA.
- 100
µg total RNA from 0.2 g fresh weight of 2-cell stages of protonema
regenerated from protoplasts.
-
RLT buffer in the kit works well for protonemata.
2)@ ISOGEN, ISOGEN-LS (NIPPON GENE,
http://nippongene.com/)
Follow
manufacturer's manual.
Note:
- This
method is as easy as method 1).@ However
the purity of total RNA from gametophores is poor and it is hard to concentrate
total RNA to more than 200ng/µl.
- This kit
is used to get total RNA without genomic DNA.@ In some cases, we extract RNA with the RNeasy
kit, then use ISOGEN to remove genomic DNA contaminants from the RNA fraction.
- We
extracted total RNA with this kit from young sporophytes with archegonium (~30
µg from 14,500 archegonium, T. Nishiyama, unpublished).
- For
protonemata: 50 µg/g fresh weight OD260/OD280 = 1.8
and for gametophore: 10 µg/g fresh weight OD260/OD280 =
1.2 (yellowish color)
- Remove
gel-like precipitates (when for protonema) or soft precipitates (gametophore)
by pipetting (avoiding the RNA pellet), when LiCl precipitation is carried
out.@ When EtOH precipitation is
performed, the translucent pellet contains RNA.
3)@ GuSCN and CTAB method
<Extraction of total RNA>@ (large scale)@ (It takes ~8 h)
1.
Buffer GnSCN@@@@@@@@@@@@@@@@@@ final conc.@@@@@@@ 15ml@@@ 30ml@@@ 50ml@@@
GuSCN
( Guanidine thiocyanate , mw=118.2) 4 M 7.1
g@@@ 14.2 g@@ 24 g
NH4SCN
( ammonium thiocyanate, mw=76.12)1 M 1.14
g@@ 2.28 g@@ 3.8
g
1
M Tris-HCl (pH 7.5)@@@@@@@@@@@@@@ 100 mM@ 1.5 ml@@ 3
ml@@@@ 5 ml
N-lauryl
sarcosine@@@@@@@@@@@@@@@@@ 1%@@@@@ 0.15 g@@ 0.3
g@@@ 0.5 g
Antifoam
A Emulsion (sigma A5758)@@ trace@@@@ 1 drop@@ 1
drop@@ 1drop
PVP
360,000@@@@@@@@@@@@@@@@@@@@@ < 0.5%@@ 0.075 g@ 0.15
g@@ 0.25 g
beta-mercaptoethanol@@@@@@@@@@@@@@@@@@@@@@@ 1%@@@@@ 150 µl@@ 300 µl@@ 500 µl
2.
CTAB buffer@@@@@@@@@@@@@@@@@@@ final conc.@@@@@@@ 50 ml@@@ 100
ml
CTAB@@@@@@@@@@@@@@@@@@@@@@@@@@ 2%@@@@@ 1 g@@@@@ 2
g
1
M Tris.HCl , pH7.5@@@@@@@@@@@@@@@@@@@@@@@ 50
mM@@ 2.5 ml@@ 5 ml
0.5
M EDTA.Na2@@@@@@@@@@@@@@@@@@ 5
mM@@@ 0.5 ml@@ 1 ml@@@@
5
M NaCl@@@@@@@@@@@@@@@@@@@@@@@@ 0.84 M@@ 8.4 ml@@ 16.8
ml
up
to @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ 50
ml@@@ 100 ml
Add
beta-mercaptoethanol just before use@@@@@@@@@@@@@@@ 500
µl@@ 1 ml
3.
10% CTAB buffer@@@@@@@@@@@@@@@@@@@@@@@ final
conc.@@@@@@@ 50 ml
CTAB@@@@@@@@@@@@@@@@@@@@@@@@@@ 10%@@@@ 5 g@@@@@
5M
NaCl@@@@@@@@@@@@@@@@@@@@@@@@ 0.7 M@@@ 7 ml@@@@@@@@@@@@
DEPC
water up to 50 ml
4.
CTAB precipitation buffer@@@@@@@@@ final
conc.@@@@@@@ 50 ml
CTAB@@@@@@@@@@@@@@@@@@@@@@@@@@ 1%@@@@@ 0.5 g
1
M Tris.HCl, pH 7-8@@@@@@@@@@@@@@@@@@@@@@@ 50
mM@@ 2.5 ml
0.5
M EDTA@@@@@@@@@@@@@@@@@@@@@ 5 mM@@@ 500 ul
DEPC
water@ up to@@@@@@@@@@@@@@@@@@@@@@@@ 50 ml
5.
High-salt TE@@@@@@@@@@@@@@@@@@@@@@@@@@@ 6.
Chloroform/Isoamylalcohol = 24 : 1
0.5
ml Tris.HCl, pH 7-8
0.1
ml 0.5 M EDTA
10
ml 5 M NaCl (final 1M)
DEPC
water to 50 ml
1.@ Grind in
liqN2 with mortar and pestle
2.@ Transfer
powder to 50 ml tube with 15 ml GuSCN B@
(5 g moss/15 ml at most)
3.@ Shake
vigorously
4.@ Add 15 ml of
C/I@ (seal with parafilm)
5.@ Shake
vigorously for 5 min.
6.@ Cfg at 6K
rpm (Hitachi himac CR20E, R12A2 rotor, #25) for 12 min. @RT
7.@ Collect
aqueous phase@@@ (should be a little
brownish)
8.@ Repeat C/I
extraction two more@ (total 3 times)
9.@ Collect
aqueous phase (ca. 14 ml)
10. Add 1/10 vol of 3 M NaOAc (pH 5.2) and 2-2.5
vol of 100% of EtOH@ (becomes cloudy)
11. Place on ice
for 30 to 60 min.
12. Cfg at 9K
rpm for 20 min. at 4˚C
13. Rinse pellet
with 80% EtOH,@@ once
14. Resuspend in
5 ml of CTAB B@ (combine tubes, 15-20
ml/tube, ideally 15 ml)*
(warm at 55˚C for 20 min,@ pipetting to <1mm pieces, then cool to RT)
(*)@ you may use falcon2059
and cfg 5k-6k (or 7k with adaptor) with angle roter #7
15. Add 5 ml of
C/I and vortex well
16. Cfg at 5K
for 10 min. at RT@@@ (thin white
interphase)
17. Harvest the
sup (~ 5 ml)
18. Add 0.6 ml
of 10% CTAB and mix well
19. Add 5.6 ml
(1 vol) of C/I, then mix
20. Cfg at 5K
for 20 min. at RT
21. Take
sup@ (sup= about 5 ml,@ almost no interphase)
22. Add 2-ME to
0.5%
@@@ Add 1.2 vol
(6ml) of CTAB pptn B@ (become cloudy)
23. Incubate at
RT for 30 to 60 min.
24. Cfg at 9K
for 15 min at RT
25. Dissolve the ppt in 5 ml High-Salt TE B (add 2-ME
at 0.5% just before use), then heat to 50-60 ˚C for 2 – 3 min@ (by pipetting)
26. Add 2.5 vol
(12.5 ml) of 100% EtOH
27. Place on ice
for 30 to 60 min.
28. Cfg at 9K
for 20 min. at 4˚C
29. Rinse with
80% EtOH once
30. Dissolve in
300 µl TE (300 µl/ a 50 ml tube)
This
resultant fraction contains total RNA and genomic DNA.@ To remove genomic DNA, we usually perform
the following protocol by using ISOGEN-LS (NIPPON GENE).@
(300 µl; convenient for ISOGEN-LS, see below)
(by pipetting and still cloudy)
<ISOGEN-LS (NIPPON GENE)>@ (2.5 h)
31. Transfer the
solution into 1.5 ml tube@ (<300
µl / a tube)
32. Add 3 vol.
of ISOGEN-LS (900 ul /tube)
33. Mix well and
incubate for 5 min at RT
34. Add 0.8 vol.
(300*0.8=240 µl) of CHCl3 (-IAA)
35. Shake
vigorously for 15 sec and then incubate for 2 to 3 min. at RT
36. Cfg at 15K
rpm for 15 min. at 4ºC
37. Collect
aqueous phase@ (about a half of the
above sum, ~750 ul)
38. Add 1 vol.
of isopropanol
39. Incubate for
10 min at RT
40. Cfg at 15K
for 10 min at 4˚C
41. Rinse with
80% EtOH, once
42. Dry
43. Dissolve in H2O or TE@ (~150 µl / 1.5 ml tube = 50 ml tube, if 5 g, 200 µg
RNA/150 µl)
44. AGE to
check@@ (loading 1ul is usually enough
even in DNA gel)
45. Quantification
by spectrophotometer@
4)
DYNABEADS mRNA DIRECT kit (DYNAL)
E
3 g fresh or frozen samples
DYNABEADS
mRNA DIRECT kit (DYNAL) or prepare the following reagents
@
EDynabeads
Oligo (dT)25 (DYNAL) Magnetic beads
E
Lysis/Bindingbuffer
100 mM Tris-HCl pH 8.0
500 mM LiCl
10 mM EDTA pH 8.0
1% SDS
5 mM DTT
E
SDS+Washingbuffer
10 mM Tris-HCl pH 8.0@@@@@
0.15 M LiCl@@@@@@
1 mM EDTA
0.1% SDS
E
Washingbuffer
10 mM Tris-HCl pH 8.0
0.15 M LiCl@@@@@@
1 mM EDTA
E
Elutionbuffer
2 mM EDTA pH 8.0
Magnetic
stand for recover magnetic beads
E Dynal MPC-1
E Dynal MPC-E-1
E Heat block
Follow
the manufacturer's instruction, protocol B-for large scale mRNA isolation
Note
As
the purity of poly(A)+ RNA obtained in this way is not good, we further purify
the RNA by ISOGEN-LS (NIPPON GENE).
Yield
and purity just after the extraction with
protonemata:
3 ug poly(A)+RNA/g fresh weight,@ OD260/OD280=1.6
gametophores:
1-2 ug poly(A)+RNA/g fresh weight,@ OD260/OD280=1.6
For total RNA including small RNAs
with miRNeasy Mini Kit (Qiagen)
Follow
manufacturer's manual.
Note:
-
The protocol for animal tissues was modified at several steps as follows:
@ 1) Grind moss samples under liquid nitrogen.
@ 2) Transfer cell powder into QIAzol Lysis
Reagent, and incubate at 54˚C for 5 min.
@ 3) Transfer QIAzol sol containing cells into
QIAshredder (not supplied in kit).
@ 4) Carefully transfer the supernatant of the
flow-through fraction to a new tube
@ 5) Additional warming is not needed.
@ 6) following steps are same as the animal
tissue protocol.
-
200 µg total RNA from 0.7 g fresh weight of dissected gametophores