John M. S. Bartlett.pdf - Bio-Nica.info

248 Ringquist et al.
5. Total RNA samples were adjusted to 400 ng/µL with distilled water, checked for the
integrity of the rRNAs by 1% agarose gel electrophoresis, and stored at –20°C.
3.2. cDNA Synthesis
1. Reverse transcription was performed on total RNA using at least three twofold dilutions of
total RNA per sample between 1000 and 125 ng per 10 µL of reaction (see Note 1). cDNA
synthesis uses oligo(dT) 20 for first strand priming, or alternatively, an arbitrary sequence
primer can be used under exactly the same conditions. The reaction mixture contained
20 µL of purified, DNase I-treated RNA plus 5 µL of 5× reverse transcription mixture.
cDNA reactions were performed at room temperature (usually 23°C) for 15 min followed
by incubation at 37°C for 1 h.
2. The reactions were stopped by heating for 5 min in a boiling water bath followed by
cooling on ice. Reactions should be diluted fourfold with 75 µL of distilled water and
stored at –20°C, or one can proceed to the next step. (see Note 2).
3.3. RAP-PCR
1. 2× RAP-PCR mixture was prepared using different pairs of arbitrary primers (see Note 3).
In Fig. 1, primers A and B were used.
2. Diluted cDNAs (10 µL) were mixed with the same volume of 2X RAP-PCR mixture.
Thermocycling was performed using an initial 3-min incubation at 94°C followed by 35
cycles of 94°C for 1 min, 35°C for 1 min, and 72°C for 2 min.
3.4. Polyacrylamide Gel Electrophoresis
1. An aliquot of the amplification products (2 µL) was mixed with 9 µL of formamide dye
solution, denatured at 85°C for 4 min, and chilled on ice. A sample of 2 µL was loaded
onto a 4% polyacrylamide, 8 M urea gel, prepared with 1× TBE buffer. The PCR products
resulting from different concentrations of the same RNA template were loaded side by
side on the gel.
2. Electrophoresis was performed at 1700 V or at a constant power of 50 to 70 W until the
xylene cyanol tracking dye reached the bottom of the gel (about 4 h). The gel was dried
under vacuum and either placed on Kodak BioMax X-Ray film for 16 to 48 h or used to
expose a phosphor screen (Molecular Dynamics, Sunnyvale, CA) and analyzed on a Storm
820 (Molecular Dynamics, Sunnyvale, CA). The results are shown in Fig. 1.
3.5. Fluorescent Labeling
1. Up to 10 µg of PCR product from the RAP-PCR can be purified using a QIAquick PCR
Purification Kit (Qiagen, Valencia, CA), which removes unincorporated bases, primers,
and primer dimer less than 40 bp. DNA was recovered in 50 µL of 10 mM Tris-HCl
(pH 8.3), usually yielding 2 to 3 µg of DNA per RAP-PCR amplification. Recovered PCR
products were quantified by spectroscopy assuming 33 µg/mL/OD unit.
2. Klenow labeling of RAP-PCR DNA was performed by random primed synthesis using up
to 1 µg of PCR product per reaction. PCR product was mixed with 12 µg total of 6-mer or
9-mer random sequence primer and boiled for 4 min, cooled to room temperature, and spun
briefly in a microfuge. The volume was adjusted to 20 µL, 25 µL of 2× Klenow reaction
mixture, and 5 µL of either 1 mM Cy3-dCTP or 1 mM Cy5-dCTP (Amersham, Piscataway,
NJ) was added and the mixture was incubated at 37°C for 4 h. The reaction was stopped by
incubation at 70°C for 10 min to inactivate the Klenow fragment. Alternatively, addition of
EDTA pH 8 to a final concentration of 10 mM can also be used. Probes from oligo (dT) n can
be prepared by standard means to examine more abundant transcripts (see Note 4).