10. Use a nanodrop to measure concentration and quality. Take
1 μg total RNA and mix with 5 μL FA buffer. Boil the mixture
at 95
C for 3 min and put it on ice for 2 min. Run 1% Agarose
gel to check quality. Normalize RNA from different samples to
the same concentration (see Note 9).
3.5 Preparation of
RNA with Radiolabeled
Marker Cocktail and
Size Selection
1. Prepare a 15% urea-polyacrylamide gel (Table 2) with the size
of 16 cmÂ16 cm Â1.5 mm (The gel volume is about 40 mL/
gel).
2. Take 1–20 μg RNA of each sample, dilute it to 20 μL, add
21 μL FA buffer and 1 μL labeled 19/24 nt marker. Mix well.
3. Denature at 95
C for 2 min and cold down on ice for 2 min.
4. Assemble cast with electrophoresis tank. Use clips to fix a piece
of aluminum pad with thin glass plate of the cast to share heat
when running electrophoresis. Fill in the top and bottom tank
with 1Â TBE buffer made with RNase-free water.
5. Use a needle and a syringe to clean wells to make sure no urea
remains. Use a curved needle and a syringe to remove bubbles
at the bottom.
6. Load samples and run electrophoresis at 500 V or 200 mA or
30 W per gel for 1 h.
7. Dismantle the gel cast and leave the gel on the top of thick glass
plate matching the labels. Wrap the gel in plastic film. Mark the
gel using small radioisotope chunks as described above. Cover
it with a phosphor screen and keep them in dark for 30 min to
1 h.
8. Expose the phosphor screen and print out the image as the
original size (Fig. 3b). Align the gel on glass to the figure. Draw
the position along the outline of the bands. Then use a clean
blade to cut the gel along the sketch. Chop that region into
small pieces and carefully transfer them to a 1.5 mL tube with
350 μL 0.4 M NaCl. Use clean water to rinse and kim wipe to
clean the blades between samples.
9. Tumble in the Eppendorf thermomixer at a speed of 1200 rpm
/min at 4
C overnight.
10. Centrifuge at 12,000 g for 5 min at 4
C. Take supernatant out
to a new tube. Add 100 μL 0.4 M NaCl to each old tube and
tumble 30 min at 4
C.
11. Centrifuge at 12,000 g for 5 min at 4
C. Mix supernatant to
previous one of each sample. Add 1 mL ethanol and 1 μL
glycogen, mix well, and incubate at À20
C overnight.
3.6 3
0 Ligation
1. Centrifuge at 21,130 g for 30 min at 4
C. Decant supernatant
and spin down again. Discard residual and air-dry pellet in a
clear area for 5 min.
Identification and Quantification of sRNAs
243
1 μg total RNA and mix with 5 μL FA buffer. Boil the mixture
at 95
C for 3 min and put it on ice for 2 min. Run 1% Agarose
gel to check quality. Normalize RNA from different samples to
the same concentration (see Note 9).
3.5 Preparation of
RNA with Radiolabeled
Marker Cocktail and
Size Selection
1. Prepare a 15% urea-polyacrylamide gel (Table 2) with the size
of 16 cmÂ16 cm Â1.5 mm (The gel volume is about 40 mL/
gel).
2. Take 1–20 μg RNA of each sample, dilute it to 20 μL, add
21 μL FA buffer and 1 μL labeled 19/24 nt marker. Mix well.
3. Denature at 95
C for 2 min and cold down on ice for 2 min.
4. Assemble cast with electrophoresis tank. Use clips to fix a piece
of aluminum pad with thin glass plate of the cast to share heat
when running electrophoresis. Fill in the top and bottom tank
with 1Â TBE buffer made with RNase-free water.
5. Use a needle and a syringe to clean wells to make sure no urea
remains. Use a curved needle and a syringe to remove bubbles
at the bottom.
6. Load samples and run electrophoresis at 500 V or 200 mA or
30 W per gel for 1 h.
7. Dismantle the gel cast and leave the gel on the top of thick glass
plate matching the labels. Wrap the gel in plastic film. Mark the
gel using small radioisotope chunks as described above. Cover
it with a phosphor screen and keep them in dark for 30 min to
1 h.
8. Expose the phosphor screen and print out the image as the
original size (Fig. 3b). Align the gel on glass to the figure. Draw
the position along the outline of the bands. Then use a clean
blade to cut the gel along the sketch. Chop that region into
small pieces and carefully transfer them to a 1.5 mL tube with
350 μL 0.4 M NaCl. Use clean water to rinse and kim wipe to
clean the blades between samples.
9. Tumble in the Eppendorf thermomixer at a speed of 1200 rpm
/min at 4
C overnight.
10. Centrifuge at 12,000 g for 5 min at 4
C. Take supernatant out
to a new tube. Add 100 μL 0.4 M NaCl to each old tube and
tumble 30 min at 4
C.
11. Centrifuge at 12,000 g for 5 min at 4
C. Mix supernatant to
previous one of each sample. Add 1 mL ethanol and 1 μL
glycogen, mix well, and incubate at À20
C overnight.
3.6 3
0 Ligation
1. Centrifuge at 21,130 g for 30 min at 4
C. Decant supernatant
and spin down again. Discard residual and air-dry pellet in a
clear area for 5 min.
Identification and Quantification of sRNAs
243