Analysis of Staphylococcus aureus Gene Expression During PMN Phagocytosis - Bacterial Pathogenesis: Methods and Protocols - page 111

RNA. Purity of RNA sample is vital for microarray analysis when working with
heterogeneous samples ( see Note 2 for RNA quantification).
3.5. cDNA Synthesis from S. aureus-PMN RNA
1. Add 10 μg of purified S. aureus -PMN RNA (can use up to 20 μL, final concen-
tration will be ˜0.33 μg/μL) to a 0.2-mL thin-walled PCR tube.
2. To the sample, add 10 μL of 75 ng/μL random primers (final concentration 25
ng/μL).
3. Add enough nuclease-free water to make the final volume of the sample 30 μL.
4. Incubate the RNA-primer mix at 70 °C for 10 min and 25 °C for 10 min, and
chill to 4 °C. Use a thermocycler for all cDNA and target labeling reactions.
5. Make a master mix of cDNA synthesis components as follows (final concentra-
tions are listed in parenthesis): 12 μL of 5× 1st Strand Buffer (1×), 6 μL of 100
m M DTT (10 m M ),3μLof10m M dNTPs (0.5 m M ), 1.5 μL SUPERase·In at 20
U/μL (0.5 U/μL), and 7.5 μL of SuperScript II (or III) at 200 U/μL (25 U/μL).
6. To the RNA-primer mix (30 μL), add 30 μL of master mix from step 5 .
7. Synthesize cDNA by PCR as follows: 25 °C for 10 min, 37 °C for 60 min, 42 °C
for 60 min. Stop reaction by inactivating SuperScript II (or III) at 70 °C for 10
min. Cool sample to 4 °C.
8. Remove RNA by adding 20 μL of 1 N NaOH to the newly synthesized cDNA
sample. Incubate at 65 °C for 30 min.
9. Neutralize the reaction by adding 20 μL of 1 N HCl.
10. Clean-up the synthesis product using a QIAquick Column (as described by the
manufacturer). Elute cDNA with 40 μL of elution buffer (Buffer EB included
in the kit) ( see Note 2 about cDNA quantification).
11. Store purified cDNA at -20 °C.
3.6. cDNA Fragmentation
1. To the 40 μL of purified cDNA (from the previous step) containing 2-10 μg, add
5 μL of 10× One-Phor-All Buffer (1× final concentration).
2. Add 0.6 U of DNase I for each μg of cDNA. Dilute DNase in 1× One-Phor-All
Buffer ( see Note 3 for information about checking the activity of the DNase I
enzyme).
3. Add nuclease-free water until the final volume reaches 50 μL.
4. To fragment the cDNA, incubate the reaction mixture at 37 °C for 10 min.
5. Stop the reaction by inactivating the DNase at 98 °C for 10 min.
6. Store the fragmented cDNA at -20 °C until use ( see Note 3 about checking
fragmented cDNA).
3.7. Labeling Fragmented cDNA with Biotin-ddUTP
1. To label fragmented cDNA, add 20 μL 5× reaction buffer, 10 μL 10× CoCl 2 ,
and 1 μL biotin-ddUTP, and 2 μL terminal deoxynucleotide transferase to 39 μL