RT-PCR analysis of RNA extracted from Bouin-fixed and paraffin-embedded lymphoid tissues.
In the present study, we have investigated whether RNA can be efficiently isolated from Bouin-fixed or formalin-fixed, paraffin-embedded lymphoid tissue specimens. To this aim, we applied a new and simple method that includes the combination of proteinase K digestion and column purification. By this method, we demonstrated that the amplification of long fragments could be accomplished after a pre-heating step before cDNA synthesis associated with the use of enzymes that work at high temperature. By means of PCR using different primers for two examined genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]- and CD40), we amplified segments of cDNA obtained by reverse transcription of the isolated RNA extracted from Bouin-fixed or formalin-fixed paraffin-embedded tissues. Amplified fragments of the expected sizes were obtained for both genes tested indicating that this method is suitable for the isolation of high-quality RNA. To explore the possibility for giving accurate real time quantitative RT-PCR results, cDNA obtained from matched frozen, Bouin-fixed and formalin-fixed neoplastic samples (two diffuse large cell lymphomas, one plasmacytoma) was tested for the following target genes: CD40, Aquaporin-3, BLIMP1, IRF4, Syndecan-1. Delta threshold cycle (DeltaC(T)) values for Bouin-fixed and formalin-fixed paraffin-embedded tissues and their correlation with those for frozen samples showed an extremely high correlation (r > 0.90) for all of the tested genes. These results show that the method of RNA extraction we propose is suitable for giving accurate real time quantitative RT-PCR results.
Gloghini
Annunziata
A
Division of Pathology, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientifico National Cancer Institute , via Pedemontana Occidentale, Aviano I-33081, Italy.
Canal
Barbara
B
Klein
Ulf
U
Dal Maso
Luigino
L
Perin
Tiziana
T
Dalla-Favera
Riccardo
R
Carbone
Antonino
A
D019342:Acetic Acid / Q000494:pharmacology; D051400:Aquaporin 3; D020346:Aquaporins / Q000378:metabolism; D001331:Automation; D019013:CD40 Antigens / Q000096:biosynthesis; D002460:Cell Line; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D005404:Fixatives / Q000494:pharmacology; D005557:Formaldehyde / Q000494:pharmacology; D006801:Humans; D008198:Lymph Nodes / Q000378:metabolism / Q000473:pathology*; D008562:Membrane Glycoproteins / Q000378:metabolism; D010232:Paraffin / Q000494:pharmacology; D016612:Paraffin Embedding; D010851:Picrates / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011509:Proteoglycans / Q000378:metabolism; D012313:RNA / Q000032:analysis* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D053668:Syndecan-1; D053667:Syndecans; D016707:Tissue Fixation
C496027:AQP3 protein, human; D020346:Aquaporins; D019013:CD40 Antigens; D017931:DNA Primers; D018076:DNA, Complementary; D005404:Fixatives; D008562:Membrane Glycoproteins; D010851:Picrates; D011509:Proteoglycans; D012333:RNA, Messenger; C508241:SDC1 protein, human; D053668:Syndecan-1; D053667:Syndecans; D051400:Aquaporin 3; D005557:Formaldehyde; C026239:Bouin's solution; D012313:RNA; D010232:Paraffin; D019342:Acetic Acid