4 Notes
1. Several hazardous chemicals are used for RNA extraction and
northern blotting:
(a) Phenol present in TRIzol is toxic and corrosive, and
should be handled under a fume hood.
(b) Chloroform is toxic and a suspected carcinogen.
(c) DEPC is a suspected carcinogen and should be handled
under a fume hood. After autoclaving, the diluted aqueous solution is safe for benchtop experiments.
(d) β-Mercaptoethanol is toxic, and should be handled under
a fume hood.
(e) Acrylamide monomer is a neurotoxin and a potential
carcinogen.
Read the material safety data sheet of each chemical for
further information.
2. The Dual Adjustable Slab Gel Kit (DASG-250-02, CBS Scientific—Fisher Scientific, NH, USA) is recommended for use
with this protocol. It includes everything needed for gel casting
and polyacrylamide gel electrophoresis (PAGE), including
glass plates (16.5 cm  17 cm), spacers, and combs
(0.75 mm with 20 wells).
3. Caution: Radiation protection measures must be taken while
handling [α
32
P]-dCTP or [γ
32
P]-ATP, and all derived
materials.
4. Alternatively, one can directly map the trimmed reads to the
reference genome using Bowtie (options: -v 1 -k 50 --best -strata --no-unal), and then perform SAM-BAM format conversion, mapped read sorting, and tallying using SAMtools.
5. Thorough grinding is essential for optimal RNA extraction.
Automated grinders (e.g., Silamat S6; Ivoclar Vivadent, Liechtenstein) are available for higher throughput applications.
Make sure that the samples never thaw during the grinding
process.
6. This protocol can be adapted to smaller amounts of input
material. In this case, freeze A. thaliana inflorescences
(or other plant tissue) in 1.5 mL safe-lock microfuge tubes
with 4–6 glass beads. Grind the samples for 8 s using a Silamat
device, and then transfer the tube back to liquid nitrogen to
avoid thawing. Repeat the grinding step a second time to
obtain a fine powder. All volumes during the RNA extraction
can thus be reduced tenfold. Centrifugation steps are performed at 15,000 Â g, and the RNA is resuspended in 50 μL
DEPC-treated Milli-Q water.
406
Marcel Bo ¨ hrer et al.
1. Several hazardous chemicals are used for RNA extraction and
northern blotting:
(a) Phenol present in TRIzol is toxic and corrosive, and
should be handled under a fume hood.
(b) Chloroform is toxic and a suspected carcinogen.
(c) DEPC is a suspected carcinogen and should be handled
under a fume hood. After autoclaving, the diluted aqueous solution is safe for benchtop experiments.
(d) β-Mercaptoethanol is toxic, and should be handled under
a fume hood.
(e) Acrylamide monomer is a neurotoxin and a potential
carcinogen.
Read the material safety data sheet of each chemical for
further information.
2. The Dual Adjustable Slab Gel Kit (DASG-250-02, CBS Scientific—Fisher Scientific, NH, USA) is recommended for use
with this protocol. It includes everything needed for gel casting
and polyacrylamide gel electrophoresis (PAGE), including
glass plates (16.5 cm  17 cm), spacers, and combs
(0.75 mm with 20 wells).
3. Caution: Radiation protection measures must be taken while
handling [α
32
P]-dCTP or [γ
32
P]-ATP, and all derived
materials.
4. Alternatively, one can directly map the trimmed reads to the
reference genome using Bowtie (options: -v 1 -k 50 --best -strata --no-unal), and then perform SAM-BAM format conversion, mapped read sorting, and tallying using SAMtools.
5. Thorough grinding is essential for optimal RNA extraction.
Automated grinders (e.g., Silamat S6; Ivoclar Vivadent, Liechtenstein) are available for higher throughput applications.
Make sure that the samples never thaw during the grinding
process.
6. This protocol can be adapted to smaller amounts of input
material. In this case, freeze A. thaliana inflorescences
(or other plant tissue) in 1.5 mL safe-lock microfuge tubes
with 4–6 glass beads. Grind the samples for 8 s using a Silamat
device, and then transfer the tube back to liquid nitrogen to
avoid thawing. Repeat the grinding step a second time to
obtain a fine powder. All volumes during the RNA extraction
can thus be reduced tenfold. Centrifugation steps are performed at 15,000 Â g, and the RNA is resuspended in 50 μL
DEPC-treated Milli-Q water.
406
Marcel Bo ¨ hrer et al.