Gene expression in formalin-fixed paraffin-embedded lymph nodes.
Elucidation of molecular pathways involved in development of human lymphoma requires efficient methods for tackling gene expression in lymph nodes. Expression studies of transcription factors in these malignancies facilitate understanding the changes occurring in neoplastic transformation and lymphoma development. Excised lymph nodes are routinely fixed in formalin and embedded in paraffin for diagnosis and stored in many hospitals' pathology archives. These tissues represent a precious resource for research since they allow retrospective studies to cover a broad range of human lymphoma even the less frequent types. Reverse transcription polymerase chain reaction (RT-PCR) is a commonly used method for gene expression analysis and a reproducible protocol for RNA isolation from lymph nodes is an inevitable requirement for these studies. However, formalin fixation and paraffin-embedding interfere with the quality of RNA especially when isolated from lymph nodes being the most fragile lymphatic tissues. We present here a simple and fast method for RNA isolation from formalin-fixed paraffin-embedded lymph nodes that can be successfully applied for RT-PCR as well as for quantitative RT-PCR analysis. We tested diverse isolation reagents and combined a range of factors in order to get a high quality RNA for retrospective studies of gene expression in human lymphoma samples. Our modified method of RNA extraction from FFPE provides superior yields and purity based on qPCR data.
Antica
Mariastefania
M
Laboratory for Electron Microscopy, Bijenicka 54, Rudjer Boskovic Institute, HR-10000 Zagreb, Croatia. antica@irb.hr
Paradzik
Mladen
M
Novak
Sanja
S
Dzebro
Sonja
S
Dominis
Marija
M
D005557:Formaldehyde / Q000737:chemistry*; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D008198:Lymph Nodes / Q000378:metabolism*; D008223:Lymphoma / Q000235:genetics; D016612:Paraffin Embedding; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D012334:RNA, Neoplasm / Q000032:analysis / Q000235:genetics; D016707:Tissue Fixation
D012333:RNA, Messenger; D012334:RNA, Neoplasm; D005557:Formaldehyde