Page 7 - BM 2011 Vol.2 No.6

Bioscience Methods
BM 2011, Vol.2, No.6 http://bm.sophiapublisher.com
- 41 -
3 Methods
3.1 Reactions of reverse transcription
The experiment was carried out using E. coli strain
JM109 and JM109 that contained the plasmid pC-SG.
The pC-SG contains 6 genes as shown in table 2. All
the strains constructed in this work were verified by
PCR using appropriate primers. The LB medium was
supplemented with 30 µg/ml kanamycin. Cells were
grown in LB at 37°C with stirring shaker (200 rpm)
overnight.
Total RNA was extracted from samples collected
during the exponential growth phase using Trizol
Reagent (Invitrogen, USA) following the manufacturer's
instructions. The RNA yield was determined by
measuring the absorbance at point of 260 nm.
To set up the ligation reaction of RNA and adapter,
1 µL 0.8 µg/µL RNA, 1 µL 1.2 pM/L TSH adapter
and 6µL ddH2O were added into a tube. After keeping
it at RT for 5 min, 1 µL 10× T4-DNA ligase buffer,
0.5 µL 350 U/µL T4-DNA ligase, and 2.5 µL ddH2O
were added in the ligation reaction. The reaction was
incubated at 16℃ for 2 h, then terminated at 75℃ water
bath for 10 min and kept on ice bath at least 2 min.
To set up the reaction of reverse transcription (RT
reaction), 0.5 µL 10 mM/L dNTP, 0.25 µL 40 U/µL
RNase inhibitor and 1µL 200 U/µL reverse transcriptase
(superscriptII from Promega) were added in the
ligation reaction. The RT reaction was incubated at
42℃ for 1h, stopped at 75 ℃ water bath for 10 min,
kept it on icebath for 2 min. 1 µL 60 U/µL RNase H
was added into the RT reaction to set up the RNase H
digestion reaction. The digestion reaction was incubated
at 30℃ for 1 h, stopped at 75℃ water bath for 10 min,
kept it on icebath for 2 min.
3.2 Hybridization reactions
To set up the hybridization reaction, 1 µL 1 µg/µL driver
RNA was add to the digestion reaction. The hybridizat-
ion reaction was performed at 94℃ for 2 min, 60℃ for
5 min, 50℃ for 15 min in a thermocycler, then kept
on icebath. To digest excessing driver RNA, 1 µL
10 mg/mL RNase A was added in the hybridization
reaction and incubated at 37℃ for 1 h.
To collect mixture of cDNA/RNA and cDNA in the
reactions, 1 volume fresh mixture of Tris-phenol:
chloroform: isoamylalcohol (25:24:1) was added in
the RNase A digestion reaction and mixed well.
Centrifuged at 8 000 rpm for 10 minutes at 4 ℃, and
transferred supernatant into a new tube, then repeated
above collecting step twice, added 2.5 volumes ice-cold
ethanol and 1/10 volume 3 M NaAc in collecting
solution. After kept the solution at -20℃ for 30 min,
centrifuged the solution at 10 000 rpm for 5 minutes at
4 ℃, having pellet air-dried for 30 min.
3.3 cDNA/RNA digestion
To set cDNA/RNA digestion reaction, dissolving the
dried pellet in 8.5 µL ddH2O, incubating at 75℃ water
bath for 10 min and keep on icebath at least 2 min.
Add 1 µL universal 10× buffer M, 0.5 µL 10 U/µL
HaveⅢ , incubating the cDNA/RNA digestion reaction
at 37 ℃ for 1.5 h, Adding 1 vol chloroform:isoamyl
alcohol and mixing well. Centrifuged at 8 000 rpm for
10 minutes at 4℃ . Transferred supernatant into a new
tube, adding 2.5 volumes ice-cold ethanol and 1/10
volume 3 M NaAc, keep for 30 min or more at -20 ℃.
Centrifuged at 10 000 rpm for 5 minutes at 4 ℃, had
pellet air-dried for 30 min.
To set the ligation reaction of the second adapter,
re-dissolving the dried pellet above in 5 µL ddH2O,
1 µL 1.2 pM/L TSH adapter. After kept the ligation
reaction at RT for 5 min, add 1 µL 10× T4-DNA ligase
buffer, 0.5 µL T4- DNA ligase, 10 µL ddH2O.
Incubated the reaction at 16℃ for 2 h, terminated the
reaction at 75℃ water bath for 10 min and kept on
icebath at least 2 min.
3.4 Amplification of tester specific cDNA
To amplify tester specific cDNA with PCR, a reaction
was set by adding 2 µL mixture of the ligation reaction
above, 2.5 µL 10× PCR buffer, 2 µL 2.5 mM/L dNTP,
1 µL 10 µM/L TSH primers for each of forward and
reverse, 16.25 µL ddH2O and 0.25 µL 5 U/µL Taq
DNA polymerase in a PCR tube. The reaction was
carried out as following denature at 95 ℃ for 30 sec，
annealing at 55℃ for 45 sec, annealing temperature
rising 0.5 ℃ for each cycle, extension at 72℃ for
2.5 min, 10 cycles for total, extension at 72℃ for 10 min
and kept at 4℃ for the last cycle. PCR products were
recovered with a gel recovery kit (Promega).