Dataset features


Application: Gene expression microarray analysis
Number of samples: 41
Release date: Aug 24 2007
Last update date: Mar 16 2012
Access: Public
Diseases: Breast Neoplasms, Neoplasms
Dataset link Comparison of RNA Amplification Techniques meeting the demands for the Expression Profiling of Clinical Cancer Samples

Experimental Protocol

To compare the methods under test we generated two pools of total RNA from breast cancer cell lines MCF7 and Hs578T, respectively, which were used for to test subsequent techniques. For each method a replication including a dye-swap hybridization was performed. Over all, non-amplifying methods were replicated two times and amplifying methods 4 times. Next, we investigated whether Ribo-SPIA and TS-PCR are suitable for amplification of rather heterogenous clinical breast cancer samples. Total RNA from (estrogen receptor) ER- (patient number 180/03) and ER+ (patient number 254/03) breast cancer samples were chosen as targets for hybridization. As tumor material was limited, only one hybridization of unamplified RNA could be performed. For Ribo-SPIA and TS-PCR (17, 19 and 26 cycles) a replicate including a dye-swap hybridization was carried out. To test test stability of TS-PCR with degraded samples, total RNA from both breast cancer samples was subjected to artificial degradation by heating to 50 degrees Celsius for 0, 25 and 50 minutes. Additionally, thyroid nodules from 3 different patients (604/05, 607/05 and 618/05) were disrupted into several pieces. TS-PCR was performed on total RNA from both breast cancer samples and different extracts from thyroid cancer subpieces, respectively. The amplification product was labeled with Cy3. Universal Reference RNA (Stratagene) was used as a common reference and labeled with Cy5.








Martin Lauss