7. RNA precipitation can also be performed overnight or over
several days. Longer precipitation times will help maximize the
yield of total RNA.
8. Somewhat larger volumes of DEPC-treated water can be used
to assure that the RNA pellet is completely resuspended. However, do not dilute the total RNA excessively if a size fractionation step is to be performed.
9. The size fractionation step can be skipped for total RNA samples rich in small RNAs, such as total RNA extracted from
A. thaliana inflorescences. In such cases, 15–30 μg total
RNA can be directly used for PAGE and northern blotting.
10. To recover the high-molecular-weight (HMW) RNAs, which
are bound to the RNeasy Midi column during size
fractionation:
(a) Centrifuge the samples for 3 min at 4500 Â g and RT to
completely dry the column.
(b) Transfer the column to a clean 15 mL tube. Add 250 μL
of DEPC-treated Milli-Q water (65
C) onto the column
for elution and incubate for 1 min.
(c) Centrifuge the samples for 3 min at 4500 Â g and RT.
(d) Put this eluted sample volume back onto the column for a
second elution.
(e) Centrifuge the samples for 3 min at 4500 Â g and RT.
(f) Quantify the RNA using a Nanodrop device and store at
À20
C (or at À80
C for a longer period of time).
If size fractionation was performed using RNeasy Mini
columns, adjust the tube size, elution volume, and centrifugation speed accordingly.
11. Caution: The pellet is not very stable and can easily be lost at
this stage.
12. Alternatively, 15–30 μg total RNA can be transferred to 1.5 mL
microfuge tubes.
13. Samples can be stored frozen in RNA-loading buffer for several
days at À20
C. These samples must then be reheated to thaw
and denature the RNA (95
C for 3 min, then kept on ice) prior
to polyacrylamide gel loading.
14. Caution: The gel should not be allowed to dry out.
15. Caution: Do not leave the gel on the UV transilluminator for
an extended period.
16. Caution: Only touch the membrane on its edges, being careful
not to scratch its surface.
Genome-Scale and Northern Blot Analyses of siRNAs
407
several days. Longer precipitation times will help maximize the
yield of total RNA.
8. Somewhat larger volumes of DEPC-treated water can be used
to assure that the RNA pellet is completely resuspended. However, do not dilute the total RNA excessively if a size fractionation step is to be performed.
9. The size fractionation step can be skipped for total RNA samples rich in small RNAs, such as total RNA extracted from
A. thaliana inflorescences. In such cases, 15–30 μg total
RNA can be directly used for PAGE and northern blotting.
10. To recover the high-molecular-weight (HMW) RNAs, which
are bound to the RNeasy Midi column during size
fractionation:
(a) Centrifuge the samples for 3 min at 4500 Â g and RT to
completely dry the column.
(b) Transfer the column to a clean 15 mL tube. Add 250 μL
of DEPC-treated Milli-Q water (65
C) onto the column
for elution and incubate for 1 min.
(c) Centrifuge the samples for 3 min at 4500 Â g and RT.
(d) Put this eluted sample volume back onto the column for a
second elution.
(e) Centrifuge the samples for 3 min at 4500 Â g and RT.
(f) Quantify the RNA using a Nanodrop device and store at
À20
C (or at À80
C for a longer period of time).
If size fractionation was performed using RNeasy Mini
columns, adjust the tube size, elution volume, and centrifugation speed accordingly.
11. Caution: The pellet is not very stable and can easily be lost at
this stage.
12. Alternatively, 15–30 μg total RNA can be transferred to 1.5 mL
microfuge tubes.
13. Samples can be stored frozen in RNA-loading buffer for several
days at À20
C. These samples must then be reheated to thaw
and denature the RNA (95
C for 3 min, then kept on ice) prior
to polyacrylamide gel loading.
14. Caution: The gel should not be allowed to dry out.
15. Caution: Do not leave the gel on the UV transilluminator for
an extended period.
16. Caution: Only touch the membrane on its edges, being careful
not to scratch its surface.
Genome-Scale and Northern Blot Analyses of siRNAs
407