Several weeks into their development, the tumors from both groups of mice were removed and subjected to DNA extraction by lysis of tissue samples.
Although the heterogeneous designs of these studies (number of sample, tumor sites, tumor stages, microdissection of tissue samples, RNA extraction and RT protocols, microarray technologies or biostatistics analyses) probably explain the diversity of the results, there is nevertheless strong motivation to develop robust molecular diagnostic tests [ 28].
Culture of plants, sampling of tissues and initial preservation of tissue samples (THK, LT); Extraction, derivatization, and instrumental analysis through and including mass spectral interpretation and metabolite identification (ALD; LWS); Data analysis related to development of the biomarker metabolite set; planning and writing the manuscript (LT).
Before extraction of DNA, tissue samples (50 100 mg) were rinsed vigorously with 1 ml of PBS and cut into fine pieces with sterile scissors.
For RNA extraction of minute tissue samples, tissues were once fixed with ethanol-acetate (3∶1) solution, and the solution subsequently substituted for PBS (Phosphate Buffered Saline) with 10% sucrose [16].
We thank M. Talamini, B. Meseck-Selchow, F. Carew-Jones and the staff at our animal facility (ZFE Frankfurt) for technical assistance, and T.P. Velavan for help with sequential extraction of mouse tissue samples.
Protein extraction of paired tissue samples (carcinoma/normal) from six nephrectomy specimens was carried out as described by Fritzsche et al. [ 9].
The total run time for extraction of 96 tissue samples was 1 h 40 min or 3 h 20 min when DNA alone or DNA and RNA, respectively, was recovered.
To support computational predictions of the damaging nature of SETD2 mutations, we assessed H3K36 trimethyltransferase activity in histone acidic extractions of patient tissue samples through Western blotting for H3K36me3 levels, an indicator of SETD2 activity [ 11].
