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

AU-Differential Display 227
DNase treatment efficiency and titration and normalization of cDNA samples were
performed by PCR as described (12).
In general, it is always easy to get amplification products. However, this does
not guarantee that those products are specific and reproducible. When the cDNA
concentration is below a minimal threshold, the final products belong to nonspecific
templates that were selected and amplified. This is why both minimal amounts of RNA
to be retrotranscribed and cDNA normalization are fundamental. The minimal amount
ensures that specific targets can be easily found and selected during the first cycles
of PCR, and the normalization ensures that DD profiles between samples activated
and resting can be compared.
The major procedures described in the protocol refer to RNA preparation and DNase
treatment, cDNA synthesis, AU-DD, and confirmation of differential expression.
2. Materials
In general, any reagent of analytical grade was considered of sufficient purity for
general procedures, such as electrophoresis or initial stages of RNA purification.
Otherwise, reagents added to enzymatic reactions or intended to dissolve RNA were of
molecular biology or ultrapure grades.
2.1. RNA Preparation
1. Lysis solution: GCS (Guanidinium thiocyanate (GuTh), Citric acid, Sarkosyl) is a
modification of Sacchi and Chomczynski solution D (14), that is, 4 M GuTh, 50 mM
sodium citrate (citric acid adjusted to pH 3.8 with NaOH), and 0.5% Sarkosyl. The solution
can be stored for up to no more than 1 mo at 4°C.
2. Double-distilled phenol (Sigma). Phenol was saturated and equilibrated in ultrapure water
and treated with 8-hydroxyquinoline (Sigma) (13) yielding unbuffered acidic phenol
(pH 4 to 5). Aliquots containing a water overlay were dispensed in sterile polypropylene
tubes and stored frozen at –20°C. After thawing, they were kept at 4°C and discarded
after 2 wk.
3. Complete Lysis solution (GCSMP): Mix GCS containing beta-2 mercaptoethanol up to 2%
with 1 volume of acidic phenol to give GCSMP immediately before use.
4. Phase lock heavy gel (PLG, Eppendorf) stored at room temperature.
5. Glycogen as coprecipitant (Roche Molecular Biochemicals) stored at –20°C.
6. Chloroformisoamyl alcohol: Chloroform should be mixed with isoamyl alcohol in a
proportion of 982 v/v (CI), respectively, in a Pyrex bottle with minimal air volume
and kept at 4°C.
7. DNAse I RQ1 from Promega at 0.2 U/µL in a solution containing 10 mM Bis-Tris-HCl,
pH 6.5, 1 mM EDTA, 5 mM MgCl 2 , 5 mM DTT, and 1 U/µL RNAsin.
8. RNasin Ribonuclease Inhibitor (40 U/µL) from Promega stored at –20°C.
9. Ethanol 96% and 75%.
10. 3 M Potassium Acetate pH 5.0 (KAc).
11. Isopropanol.
12. Guanidinium thiocyanate (4 M) in water.
2.2. cDNA Synthesis and Further Treatment
1. Reverse transcriptase: SuperScript II RNase H- (Gibco-BRL). Store at –20°C.
2. dNTPs (Amersham Biosciences) at 10 mM each.
3. RNasin Ribonuclease Inhibitor 40 U/µL from Promega (Madison, WI) stored at –20°C.