Array-based approaches to bacterial transcriptome analysis - Methods in Microbiology - page 159

manufacturer's instructions ( Figure 6.1 ). During the past decade, the Agilent Bioa-
nalyzer has become the standard tool for assessing RNA quality, particularly for
RNA from eukaryotic cells where, in addition to the electropherograms, RIN
(RNA Integrity Number) values can be used for an objective and standardized
evaluation of RNA quality.

3.2 Synthesis and labelling of cDNA
In typical array-based bacterial transcriptome studies, total RNA is converted into
first-strand cDNA using random primers and reverse transcriptase. Depending on
the scanning technology employed, cDNA is labelled either with a fluorescent
dye or with biotin (Affymetrix GeneChip platform). For fluorescence labelling,
two basic principles can be applied either direct labelling (reverse transcription with
concomitant incorporation of a cyanine-dye-coupled nucleotide) or indirect labelling
via aminoallyl nucleotides (incorporation of an aminoallyl-modified nucleotide and
subsequent chemical coupling to a cyanine dye). Possible disadvantages of direct
labelling procedures such as lower cDNA yields and less efficient incorporation
of labelled nucleotides have been discussed ( Richter et al. , 2002 ) but cannot be con-
firmed on the basis of our current experience. Before being used for hybridization,

Protocol 1 GENERATION AND LABELLING OF STRAND-SPECIFIC CDNA
FROM PROKARYOTIC TOTAL RNA
Specific Reagents: FairPlay III Microarray Labeling Kit (Stratagene) fromAgilent Technologies,
Actinomycin D (1 set¼20200 mg) from Calbiochem, CyDye Cy3 Mono-Reactive Dye 5-Pack
from GE Healthcare
Step 1. First-Strand cDNA Synthesis
1. Prepare Actinomycin D by dissolving 200 mg (entire vial) in 400 ml RNase/DNase-
free water. Make aliquots of 20 ml and store at 20 C. These aliquots are stable for
at most a month at 20 C.
2. Assemble the following components in 0.2-ml thin-walled PCR tubes: 9 ml sample
(10 mg RNA) and 1 ml Random Primer (9 mers, 500 ng/ml).
3. Heat-denature sample in a thermocycler at 70 C for 10 min. Quick-chill in an
ice-water bath for 5 min.
4. Prepare the following first-strand Master Mix according to the number of reactions
(volumes per sample):

10 AffinityScript RT buffer 2 ml

20 dNTP mix 1 ml

0.1 M DTT 1.5 ml

RNase Block (40 U/ml) 0.5 ml

Actinomycin D (500 m g/ml) 1.6 m l

RNase/DNase-free water 0.4 m l

Total 7 m l