3. Add 8 mL of TRIzol to each tube (ensure that the liquid
nitrogen has evaporated from the powder before adding the
reagent).
4. Vortex tubes until the powder melts and is homogeneously
suspended in TRIzol. When extracting many samples in parallel, the homogenized samples can be left on ice.
5. Add 1.6 mL chloroform, vortex for ~40 s, and then leave tubes
on ice for 3 min.
6. Centrifuge the samples for 15 min at 8500 Â g and 4
C.
7. Transfer the aqueous phase to a clean 13 mL roundbottomed tube.
8. Add 7 mL of cold isopropanol to each tube and mix by inverting the tube ten times.
9. Let the total RNA precipitate at À20
C for at least 2 h (see
Note 7).
10. Centrifuge the samples for 30 min at 9200 Â g and 4
C, and
then discard the supernatant.
11. Add 10 mL cold 75% ethanol, centrifuge for 15 min at
4500 Â g and 4
C, and then carefully discard the supernatant.
12. Repeat ethanol wash (step 11) to further purify the RNA.
13. Air-dry the RNA pellets, tubes upside down, for about 20 min
at RT.
14. Resuspend the RNA pellets in 120 μL DEPC-treated Milli-Q
water, preheated to 65
C (see Note 8).
15. Quantify the total RNA using a Nanodrop spectrophotometer
(absorbance at 260 nm).
16. Store the samples at À20
C for up to 1 month, or for longer
periods at À80
C.
3.3 Size
Fractionation of Total
RNA
Northern blot detection of 24 nt siRNAs can be difficult, especially
if the input total RNA is isolated from tissues like B. distachyon
leaves, which contain low levels of siRNAs compared to RNA
samples isolated from A. thaliana inflorescences. RNA size fractionation enriches for low-molecular-weight (LMW) RNAs and
thereby enhances siRNA detection (see Note 9). RNeasy Midi
columns are used here to separate LMW RNAs from highmolecular-weight (HMW) RNAs, following the “RNA cleanup”
protocol [64, 65]. HMW RNAs bind to the silica membrane of the
columns, whereas <200 nt LMW RNAs pass through and can be
recovered in flow-through and wash aliquots.
1. Transfer up to 1 mg of total RNA in a volume of 500 μL
DEPC-treated water to a 13 mL round-bottomed tube.
2. Add 2 mL of RLT buffer complemented with β-mercaptoethanol
(10 μL/mL RLT) to each sample; if RNeasy Mini columns are
used here, then adjust all volumes accordingly.
398
Marcel Bo ¨ hrer et al.
nitrogen has evaporated from the powder before adding the
reagent).
4. Vortex tubes until the powder melts and is homogeneously
suspended in TRIzol. When extracting many samples in parallel, the homogenized samples can be left on ice.
5. Add 1.6 mL chloroform, vortex for ~40 s, and then leave tubes
on ice for 3 min.
6. Centrifuge the samples for 15 min at 8500 Â g and 4
C.
7. Transfer the aqueous phase to a clean 13 mL roundbottomed tube.
8. Add 7 mL of cold isopropanol to each tube and mix by inverting the tube ten times.
9. Let the total RNA precipitate at À20
C for at least 2 h (see
Note 7).
10. Centrifuge the samples for 30 min at 9200 Â g and 4
C, and
then discard the supernatant.
11. Add 10 mL cold 75% ethanol, centrifuge for 15 min at
4500 Â g and 4
C, and then carefully discard the supernatant.
12. Repeat ethanol wash (step 11) to further purify the RNA.
13. Air-dry the RNA pellets, tubes upside down, for about 20 min
at RT.
14. Resuspend the RNA pellets in 120 μL DEPC-treated Milli-Q
water, preheated to 65
C (see Note 8).
15. Quantify the total RNA using a Nanodrop spectrophotometer
(absorbance at 260 nm).
16. Store the samples at À20
C for up to 1 month, or for longer
periods at À80
C.
3.3 Size
Fractionation of Total
RNA
Northern blot detection of 24 nt siRNAs can be difficult, especially
if the input total RNA is isolated from tissues like B. distachyon
leaves, which contain low levels of siRNAs compared to RNA
samples isolated from A. thaliana inflorescences. RNA size fractionation enriches for low-molecular-weight (LMW) RNAs and
thereby enhances siRNA detection (see Note 9). RNeasy Midi
columns are used here to separate LMW RNAs from highmolecular-weight (HMW) RNAs, following the “RNA cleanup”
protocol [64, 65]. HMW RNAs bind to the silica membrane of the
columns, whereas <200 nt LMW RNAs pass through and can be
recovered in flow-through and wash aliquots.
1. Transfer up to 1 mg of total RNA in a volume of 500 μL
DEPC-treated water to a 13 mL round-bottomed tube.
2. Add 2 mL of RLT buffer complemented with β-mercaptoethanol
(10 μL/mL RLT) to each sample; if RNeasy Mini columns are
used here, then adjust all volumes accordingly.
398
Marcel Bo ¨ hrer et al.