Comparative expression of a set of genes to an internal housekeeping control in CDNA amplified and not amplified by polyAPCR in non-hodgkins lymphoma samples obtained from fine-needle aspiration cytology
Corbi F.C.,Hematology and Hemotherapy Service |
Inaoka R.J.,Hematology and Hemotherapy Service |
Felix R.S.,Hematology and Hemotherapy Service |
Andrade V.C.C.,Hematology and Hemotherapy Service |
And 4 more authors.
Diagnostic Molecular Pathology | Year: 2010
Aim: We aimed to evaluate the amount and quality of the RNA obtained from lymph nodes of non-Hodgkin lymphomas (NHLs) patients using fine-needle aspiration cytology (FNAC), and to develop strategies to overcome eventual technical drawbacks. Materials and Methods: Twenty-six patients with NHL and 10 tonsils from children submitted to tonsillectomy underwent FNAC. The aspirates were performed using both cytoaspirator (sample A) and syringe and needle (sample B). The RNA was extracted using Trizol reagent and transcribed with the Superscript kit (Invitrogen). The quality of RNA was verified through the amplification of a β-actin 155-bp fragment. Results: Fifty-two NHL and 20 tonsil samples were analyzed. The total amount of RNA in the tonsil samples varied from <1.0 to 6.2μg with cytoaspirator (A) and from <1.0 to 4.7μg with syringe and needle (B). The total amount of RNA obtained from NHL varied from <1.0 to 6.5μg with cytoaspirator (A) and <1.0 to 5.5μg with syringe and needle. In an attempt to increase the amounts of RNA in each sample, we standardized the polyAPCR technique, which increased by 10 times the amount of cDNA in most of the test and control samples. The efficiency of the reaction was verified through the amplification of β-actin, in which 100% of the test and control samples were amplified. When polyAPCR cDNA and nonamplified cDNA samples were paired to be evaluated by real-time PCR, using glyceraldehyde-3-phosphate dehydrogenase as the constitutive gene and nuclear factor-kappa B and NFκBIA as target genes, there was equivalence in the amplifications of 100% of the 15 evaluated samples. Conclusions: Our results showed that FNAC, obtained either by cytoaspirator or syringe and needle, is a good source of small amounts of RNA. The polyAPCR technique significantly increased the amount of genomic material, which might be a cDNA source for future gene expression studies. Copyright © 2010 by Lippincott Williams & Wilkins.