Comparative expression of a set of genes to an internal housekeeping control in CDNA amplified and not amplified by PolyAPCR in non-Hodgkin's lymphoma samples obtained from fine-needle aspiration cytology.
OBJECTIVE
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.


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 beta-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 6.2 microg with cytoaspirator (A) and from <1.0 to 4.7 microg with syringe and needle (B). The total amount of RNA obtained from NHL varied from <1.0 to 6.5 microg with cytoaspirator (A) and <1.0 to 5.5 microg 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 beta-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 NFkappaBIA 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.
Corbi
Fernanda Cristina
FC
Hematology and Hemotherapy Service, Department of Pathology, Federal University of São Paulo, Vila Clementino, São Paulo, Brazil.
Inaoka
Riguel Jun
RJ
Felix
Roberta Spetic
RS
Andrade
Valéria C C
VC
Etto
Leina Yukari
LY
Vettore
André L
AL
Franco
Marcello F
MF
Colleoni
Gisele W B
GW
D000199:Actins / Q000235:genetics; D044963:Biopsy, Fine-Needle; D002648:Child; D002675:Child, Preschool; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D006801:Humans; D008198:Lymph Nodes / Q000473:pathology*; D008214:Lymphocytes; D008228:Lymphoma, Non-Hodgkin / Q000473:pathology*; D057089:Pathology, Molecular / Q000379:methods* / Q000592:standards; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000235:genetics / Q000302:isolation & purification*
D000199:Actins; D018076:DNA, Complementary; D012313:RNA