Amplification of total RNA from minute amounts of RNA of the thyroid gland for validation of molecular classifiers

A28 Fine-needle aspiration biopsy (FNAB) is the gold standard for preoperative diagnostic test of malignant thyroid nodules. However, this procedure is not capable of discriminating between adenomas and follicular carcinomas. Suspicious nodules are usually removed surgically and it requires inspection of the entire capsule in order to differentiate a follicular carcinoma from an adenoma. A precise and reliable method for distinguishing between benign and malignant thyroid nodules prior to surgery may help to reduce the number and extent of thyroidectomies. Data from gene expression analysis of postsurgical samples of adenoma and follicular carcinoma obtained by cDNA microarrays assays were submitted to Fisher linear discriminant analysis and expression signatures of individual samples of adenomas and follicular carcinomas were used to identify molecular classifiers that precisely distinguish between malignant and nonmalignant lesions. Fourteen trios of genes were able to perform the correct classification of samples. The robustness of these trios was verified by using leave-1-out cross-validation and bootstrap analyses. In order to validate this test using an independent set of samples we are current applying two strategies. First, we used FNAB of nodules after surgical removal of the gland (58 samples), in order to establish a reliable RNA amplification protocol starting from minute amounts of RNA. Second, we used material derived FNAB from patients with suspicious lesions (20 samples) representing normal thyroid, adenomas, papillary carcinomas, and follicular carcinomas. The latter procedure was approved by our Institutional Review Board and no extra manipulation of the patient was necessary. Needles that were used in diagnostic FNAB were washed with guanidinum thiocyanate-phenol solution before being discarded and these washings were used for RNA extraction. Considering the variability in cellularity from these samples, it was necessary to adapt our protocol for RNA extraction. Total RNA was extracted from all types of samples, including those with low cellularity. Samples were submitted to one round of amplification, according to a protocol previously published by our group (Gomes et al, 2003), starting from as little as 20ng of total RNA. Amplification efficiency ranged from 258 to 300 fold. A second round of amplification will be done and results of validation using these independent samples will be presented.