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

240 Khalturin, Kuznetsov, and Bosch
3.4. Electrophoresis
1. Before electrophoresis, add 7 µL of sample buffer (95% formamide, 0.25% dextran blue,
10 mM EDTA) to each 10-µL sample.
2. Denature amplified labeled fragments at 95°C for 3 min and load onto a high-resolution
denaturing polyacrylamide gel (HR-1000 4.5% matrix, Genomyx, Foster City, CA).
3. Perform the electrophoresis for 2.5 h in parallel using the GenomyxLR DNA sequencer
(according to the manufacturer’s instructions).
3.5. Detection of PCR Products, Elution, and Cloning
1. cDNA bands are detected using the GenomyxSC Fluorescent Imaging Scanner (Genomyx).
A typical gel with several differentially expressed transcripts of the freshwater polyp
Hydra vulgaris is shown in Fig. 2 (filled and open arrows). Fluorescently labeled cDNA
fragments usually range from about 100 to 2000 bp.
2. For further characterization, DNA from differentially expressed bands is localized using
the grid coordinate system provided with the GenomyxSC Fluorescent Imaging Scanner.
After excision from the gel, cDNA in gel slices can easily be recovered and reamplified
using described procedures (7,14).
4. Notes
In our minds, the most crucial steps for performing a successful differential display
screening are as follows: (1) quality of the initial RNA taken for the cDNA preparation;
(2) the quantity of cDNA used for the DD-PCR; and (3) the PCR cycling profile, which
depends greatly on the type of thermal cycler used.
1. In our experience DD-PCR works equally well in using either total RNA or mRNA. Kits
for the isolation of poly(A) + mRNA, however, seem to be more reliable. The reason is that
when total RNA is extracted, DNAse I treatment is usually needed to eliminate the DNA.
However, this procedure sometimes can lead to considerable loss of mRNA.
If extraction of total RNA is the method of choice, it is crucial to check the quality of the
RNA obtained not only by the OD 260 and OD 280 measurement but also by electrophoresis
of the RNA in an agarose gel. In the ethidium bromide-stained gel, the total RNA should
appear as a smear of approx 500 to 2000 bp with two prominent bands of 28S rRNA and
18S rRNA. If any RNA degradation or traces of high molecular weight DNA are observed,
the RNA samples should not be used in DD-PCR.
2. We prefer to synthesize cDNA using T (12) NN tailing primers (where NN stands for one of
the 12 possible combinations of 4 nucleotides) instead of standard NotI primer. Using the
same tailing primer both in reverse transcription and DD-PCR gives more distinct bands in
comparison with the case when cDNA is made with NotI primer and then one of T (12) NN
primers is used in a DD-PCR. A slight increase in the reaction temperature (40°C instead
of the usual 37°C) during reverse transcription improves the specificity of cDNA synthesis.
DD-PCR banding patterns obtained with the same random primer, but two different tailing
primers (e.g., –5′-T 12 CA-3′ and 5′-T 12 AC-3′) should be considerably different.
3. High final concentrations of dNTP (from 25 to 250 µM of each) and Mg 2+ (from 1.5 to
4 mM) may help increasing the quantity and quality of bands. For optimal results, it is
always necessary to try different dilutions of the cDNA sample (e.g., 125 → 150). A
low final concentration of cDNA in DD-PCR can lead to nonreproducible banding patterns
and even to differences within pares of tubes containing identical cDNA templates. One
typical problem encountered when doing DD-PCR is that the tailing primer does not
anneal during PCR, resulting in fragments flanked by random primer only. In that case,