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

234 Dominguez et al.
3. If the container is not a polypropylene (PPN) tube, transfer lysates to a PPN centrifuge tube.
Choose the tube so that the lysate volume is not larger than one third of its capacity.
4. Because lower RNA amounts negatively affect the reliability of DD techniques, not less
than 3 µg are processed.
5. The RNA concentration in the storage ethanol solution is one fourth of the original
concentration before adding the alcohol. In absence of salts, this solution forms a relatively
uniform suspension of RNA, which is easy to pipet. To recover an aliquot, transfer the
volume that contains the desired amount to a fresh tube. Supplement and mix with both
coprecipitant (glycogen) and 0.1 vol of KAc, pH 5.0. Store an additional hour at –80°C
and gather the sediment by centrifugation at 12,000g. After a single wash in 75% ethanol,
the pellet is ready for any downstream application.
6. To assure a complete removal of contaminating DNA, trial tests were performed. RNA
from human thymus, which copurifies with relatively high content of DNA, was used for
these assays. The best conditions were found when RNA (substrate) and DNase (enzyme)
were kept as described.
7. The absence of residual contaminating DNA was demonstrated by the failure to amplify
the genomic locus of interferon-α, a multicopy gene, by PCR. A program of 40 cycles with
an annealing temperature of 60°C was performed, with primers at 1 µM and reaction in
10 µL (primers in Table 1). The inclusion of negative controls to test for PCR contamination
is advisable. If DNA is detected, the DNAse-treated RNA sample is discarded.
8. After DNase treatment, ethanol precipitation is apparently enough to inactivate any
significant DNase activity without the need of any further treatment such as phenol.
9. The RNA pellet can be stored indefinitely in this wash solution of 75% ethanol. It is
left there before reverse transcription until it is confirmed by PCR that no residual DNA
contamination has survived (see Note 7).
10. Total RNA was always used because poly A selection from low amounts (tens of micrograms)
of RNA was found inappropriate. The overall losses were significant, and an
unquantifiable amount made the DD unreproducible and difficult to normalize.
11. Given the 3′ location of the AU motifs there was a special interest in retrotranscribing true
3′ ends. The separate step of annealing at room temperature was found to reduce primer
extensions from false poly A tails.
12. In this setting, the 1500 dilution always gives comparable results among samples; 4 µL
checked by electrophoresis usually contains 5 to 10 ng of GAPDH amplimer.
13. Because it is of interest to sample as many genes as possible different conditions are used
side by side to increase throughput. Limited sample availability can also benefit from
these variations. Primers of the series G7AU2 (Table 1) are used on cDNAs initiated
from G7AU2dT. Similarly, GTGAU2 primers are more efficient on cDNA primed from
GTGAU2dT. Both types of primers give different profiles, and different designs of the
nonspecific 5′ domain also give different patterns. Proposed variations of conditions for a
given cDNA to yield different profiles are as follows: (1) use the five primers of any series
in 40 cycles of a single round of PCR; (2) run first a nonradioactive PCR of 30 cycles with
either G7AU2 or GTGAU2, then dilute products 150 and run a series of nested PCR of
30 cycles with the remaining four inner primers (G7AU2N or GTGAU2N) in four separate
radioactive reactions; (3) reduce the concentration of cold dNTPs; (4) change the reaction
buffer (different profiles can be achieved just by changing the Mg concentration in 1 mM
differences); (5) combine any of these conditions.
14. A cold dNTP concentration of 0.2 mM on radioactive reactions gives stronger signals and
lower background than lower concentrations.