4. Add 1 μL RNase inhibitor and 1 μL SSII to each sample. Tap to
mix well. Centrifuge at 5000 g for 1 min. Incubate samples at
42
C for 1.5 h.
5. Add 40 μL 150 mM KOH/20 mM Tris-Base to each sample.
Mix well. Incubate samples at 90
C for 10 min.
6. Neutralize pH value of each cDNA sample with a certain
volume of 150 mM HCl to about 7–8 using Hydrion S/R
Dispenser pH papers.
3.9 PCR
1. Prepare Mastermix for a small-scale PCR for one sample (8 μL
10 Â KOD Buffer, 3.2 μL 25 mM MgSO 4 , 3.2 μL dNTPs
(2 mM each), 0.8 μL 5
0 primer (SR primer containing P5
sequence), 0.8 μL 3
0 primer (different index attached with P7
sequence), 8 μL cDNA, 1.6 μL KOD, and 54.4 μL water). The
total volume is 80 μL. Aliquot into 4 reactions evenly. Repeat
for other samples with the replacement of cDNA and
corresponding index primers.
2. Program PCR reactions as below: 95
C 2 min first, then
program cycles containing 95
C 20 s, 55
C 30 s, and 68
C
30 s. The cycle numbers are 12, 15, 18, and 21, matching to
each reaction above.
3. Run a 2.5% agarose gel. Put 4 products from one sample
together. Select a cycle in which the amplification is at an
exponential stage. The expected band size should be between
100 bp and 200 bp (Fig. 4a).
4. After defining the cycle numbers, prepare large-scale PCR
reaction system: 30 μL 10Â KOD Buffer, 12 μL 25 mM
MgSO 4 , 12 μL dNTPs (2 mM each), 3 μL 3
0 primer, 3 μL 5
0
primer, 30 μL cDNA, 6 μL KOD, and 204 μL water. The total
volume is 300 μL. Aliquot each reaction into 8 PCR tubes. Run
PCR program as described in step 2 but with the
corresponding cycle for each sample.
5. After amplification, combine PCR products from the same
sample to a 1.5 mL Eppendorf tube. Add 300 μL phenol:
chloroform:isoamyl alcohol (25:24:1) pH 8.0 to each tube.
Vortex for 20 s. Centrifuge at full speed at 4
C for 10 min.
6. Take the upper, aqueous phase to a new 1.5 mL tube. Add
300 μL chloroform to the supernatant and vortex for 20 s.
Centrifuge at full speed at 4
C for 10 min.
7. Decant 250 μL upper, aqueous phase to a new 1.5 mL tube
without touching the wall of the tube. Add 25 μL 3 M NaAc
and 750 μL ethanol. Mix well by rotating tubes upside down.
Then, incubate tubes at –20
C overnight.
Identification and Quantification of sRNAs
245
mix well. Centrifuge at 5000 g for 1 min. Incubate samples at
42
C for 1.5 h.
5. Add 40 μL 150 mM KOH/20 mM Tris-Base to each sample.
Mix well. Incubate samples at 90
C for 10 min.
6. Neutralize pH value of each cDNA sample with a certain
volume of 150 mM HCl to about 7–8 using Hydrion S/R
Dispenser pH papers.
3.9 PCR
1. Prepare Mastermix for a small-scale PCR for one sample (8 μL
10 Â KOD Buffer, 3.2 μL 25 mM MgSO 4 , 3.2 μL dNTPs
(2 mM each), 0.8 μL 5
0 primer (SR primer containing P5
sequence), 0.8 μL 3
0 primer (different index attached with P7
sequence), 8 μL cDNA, 1.6 μL KOD, and 54.4 μL water). The
total volume is 80 μL. Aliquot into 4 reactions evenly. Repeat
for other samples with the replacement of cDNA and
corresponding index primers.
2. Program PCR reactions as below: 95
C 2 min first, then
program cycles containing 95
C 20 s, 55
C 30 s, and 68
C
30 s. The cycle numbers are 12, 15, 18, and 21, matching to
each reaction above.
3. Run a 2.5% agarose gel. Put 4 products from one sample
together. Select a cycle in which the amplification is at an
exponential stage. The expected band size should be between
100 bp and 200 bp (Fig. 4a).
4. After defining the cycle numbers, prepare large-scale PCR
reaction system: 30 μL 10Â KOD Buffer, 12 μL 25 mM
MgSO 4 , 12 μL dNTPs (2 mM each), 3 μL 3
0 primer, 3 μL 5
0
primer, 30 μL cDNA, 6 μL KOD, and 204 μL water. The total
volume is 300 μL. Aliquot each reaction into 8 PCR tubes. Run
PCR program as described in step 2 but with the
corresponding cycle for each sample.
5. After amplification, combine PCR products from the same
sample to a 1.5 mL Eppendorf tube. Add 300 μL phenol:
chloroform:isoamyl alcohol (25:24:1) pH 8.0 to each tube.
Vortex for 20 s. Centrifuge at full speed at 4
C for 10 min.
6. Take the upper, aqueous phase to a new 1.5 mL tube. Add
300 μL chloroform to the supernatant and vortex for 20 s.
Centrifuge at full speed at 4
C for 10 min.
7. Decant 250 μL upper, aqueous phase to a new 1.5 mL tube
without touching the wall of the tube. Add 25 μL 3 M NaAc
and 750 μL ethanol. Mix well by rotating tubes upside down.
Then, incubate tubes at –20
C overnight.
Identification and Quantification of sRNAs
245