FastLane Cell cDNA Kit (50)

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Follow manufacturer's instructions

Publication protocol

The NCI-60 cell lines from 7 tissues of origin- A549 (lung, ATCC CCL185), HCT116 (colon, ATCC CCL247, gift from Prof. Sim Kim Ping), OVCAR8 (ovarian, gift from Prof. Jean Paul Thiery [24]), 786–0 (renal, ATCC CRL1932, gift from Dr. Deng Lih Wen), M14 (melanoma, gift from Prof. Marie-Véronique Clément [25]), PC3 (prostate, ATCC CRL1435), U251 (central nervous system, ATCC 09063001) were grown in Dulbecco's minimum essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (PS, Sigma). All cells were cultured in incubator with 5% CO2 humidified atmosphere at 37°C. The 7 cell lines in a 96-well plate (2×104 cells per well, biological quadruplicates) were lysed with RealTime Ready Cell Lysis Kit (Roche), FastLane Cell cDNA Kit (Qiagen), Buffer of Cell Lysate for Reverse Transcription (Signosis BioSignal Capture), iScript™ RT-qPCR Sample Preparation Reagent (Bio-Rad Laboratories), or MicroRNA Cells-to-CT™ Kit (ABI Ambion) according to manufacturer recommendation. In-house cell lysis reagent [26], [27] was modified for detection of mRNA. This reagent contained 2% Triton X-100 and 2% NP40, 1/25 of RNAsecure (ABI) and 1 unit/µl of RQ1 DNAse (Promega, MA). Cells were washed once with PBS, and incubated with the in-house lysis buffer for 5 min at room temperature. The cell lysates were transferred to PCR strip tubes and incubated for 75°C for 5 min.

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