Download side

Ethanol (Absolute, Aaper Alcohol)
Random hexamer primer (1 μg/μl, Roche Diagnostics)
Microcentrifuge tubes (nuclease-free)
Heating block or water bath preset to 16◦ C, 37◦ C, 42◦ C, 65◦ C, 70◦ C, 95◦ C
Concentrator/evaporator
Vortex
Perform first-round RNA amplification
1. For each reaction, mix the following components in a nuclease-free microcentrifuge
tube:
0.5 μg/μl T7-oligo(dT) primer
total RNA extracted from LM sample
H2 O (DEPC-treated)
1 μl
20 to 100 ng
to 10.5 μl.
2. Incubate the samples 10 min at 65◦ C and cool on ice for 5 to 10 min.
The primer anneals to poly(A)-containing RNA during this step, and thereby attaches a
copy of the T7 RNA polymerase promoter sequence to the cDNA molecules that will be
synthesized in the following steps
3. Collect the samples by “quick-spin” centrifugation 30 sec at 600 × g.
4. Add 8.5 μl of the following mixture to each tube:
10 mM dNTP mix
5× first-strand buffer
0.1 M DTT
40 U/μl RNAseOUT
5 μg/μl T4 gene 32 protein
1 μl
4 μl
2 μl
1 μl
0.5 μl.
NOTE: When preparing a cocktail master mixture to accommodate multiple samples,
excess reagents should be prepared to compensate for reagent loss during pipetting.
RNaseOUT is an inhibitor of RNaseA, RNaseB, and RNaseC type ribonucleases, and is
added to help prevent degradation of RNA samples during the ensuing reverse transcriptase reaction.
5. Mix gently and add 1 μl of Superscript II (200 U/μl) to each tube.
6. Incubate 1 hr at 42◦ C. If necessary, at this point the samples can be stored indefinitely
at –20◦ C.
Reverse-transcription of the RNA occurs during this step to form single-stranded DNA
that contains the T7 RNA polymerase promoter at 5 end.
7. Add 130 μl of the following mixture to each 20-μl reaction:
10× E. coli DNA polymerase I buffer
10 mM dNTP mix
260 μM β-NAD+
10 U/μl E. coli DNA polymerase I
2 U/μl RNase H
10 U/μl E. coli DNA ligase
H2 O
15 μl
3 μl
15 μl
4 μl
1 μl
1 μl
91 μl.
NOTE: Prepare excess reagent mixture to compensate for reagent loss during pipetting.
Laser
Microdissection
and Amplification
of Plant RNA
25A.3.8
Supplement 87
8. Mix gently and incubate 2 hr at 16◦ C.
During this step, DNA polymerase I synthesizes second-strand DNA molecules while DNA
ligase will ligate the new molecules into a single, uninterrupted DNA strand.
Current Protocols in Molecular Biology