However, after infection, all samples continue to synthesize some, although reduced, free α-globin chains, indicating that the excess of α-globin content was not completely abolished.
Five hundred ng of total RNA from 3 colon cancer tissues for the test samples and normal colon tissue for the reference samples were used to synthesize labeled cRNA (Low RNA Input Linear Amp Kit, Agilent Technologies) in the presence of cyanine 3-dCTP and cyanine 5-dCTP (Perkin-Elmer Life Sciences, Boston, MA), respectively.
Briefly, RNA (15 µg per reaction) from target (BALF-incubated bacteria) and reference (BHI-incubated bacteria) samples was used to synthesize cDNA in the presence of amino-allyl-dUTP (Sigma-Aldrich, St . Louis MO US), random octamer primers (Biocorps, Montreal, QC, Canada), and SuperScript II transcriptase (Invitrogen, Carlsbad, CA, US).
Equal amounts of environmental DNase I-treated RNA samples were used to synthesize cDNA with Superscript II Reverse Transcriptase (Invitrogen Life Technologies) at 42°C for 50 min. The reactions were treated for 15 min at 70°C to inactivate the enzyme.
Briefly, 0.5 µg of total RNA from each samples was used to synthesize fluorescent-labelled cRNA using Cyanine 3-CTP (CY3c) as described in the manual (Agilent Quick Amp Labeling Kit, one-color).
To overcome this problem, mRNA enriched samples were used to synthesize cDNA for microarray analysis and in vitro synthesized RNA was added in equal quantities to each sample (spiked in controls) for data normalization, as described elsewhere [ 44], to allow the comparison of samples from different growth stages on the same microarray.
For mRNA expression analysis, 100 ng of total RNA (250 ng for FFPE samples) were used to synthesize cDNA with High Capacity cDNA Reverse Transcription kit (Applied Biosystems), and RT-qPCR was performed using specific TaqMan assays (Applied Biosystems) for DICER (ID 00998578), DROSHA (ID 01095029), TARBP2 (ID 00998379), DGCR8 (ID 00987089) and PACT (ID 00269379).
From a training set of 18 tumor samples (eight control and 10 TF), total RNA was extracted from each sample and used to synthesize the corresponding complex probe to be hybridized on the cDNA arrays.
For Sox9 ChIP anti-Sox9 antibody (R&D Systems, AF3075) was used for immunoprecipitation. 10-15 ng of purified ChIP DNA from each sample was used to synthesize the sequencing library as instructed by the ChIP-Seq DNA sample Prep Kit (Illumina).
