Amplification of mRNA from laser-microdissected single or clustered cells in formalin-fixed and paraffin-embedded tissues for application in quantitative real-time PCR.
The determination of marker genes and gene clusters involved in disease pathogenesis is increasingly contingent on high-throughput methods of gene expression profiling. However, the concurrently increasing application of mRNA from formalin-fixed and paraffin-embedded (FFPE) tissue archives, as well as cell-type-specific approaches by laser-assisted microdissection, frequently results in very small and degraded quantities of RNA. Therefore, a successful amplification of cell-type-specific mRNA targets from FFPE tissues becomes more and more essential. To optimize the hitherto limited technical options, we applied 3 commercial amplification kits on FFPE single cells. We thereby determined the approach of target-specific cDNA amplification as being notably appropriate for subsequent real-time polymerase chain reaction, as a constant decrease of CT values by 14 polymerase chain reaction cycles could be demonstrated.
Theophile
Katharina
K
Institute of Pathology, Hannover Medical School, Hannover, Germany.
Jonigk
Danny
D
Kreipe
Hans
H
Bock
Oliver
O
D001854:Bone Marrow Cells / Q000737:chemistry / Q000473:pathology; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D005784:Gene Amplification; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007834:Lasers; D042282:Microdissection; D009369:Neoplasms / Q000737:chemistry / Q000235:genetics; D016612:Paraffin Embedding; D012333:RNA, Messenger / Q000235:genetics*; D012334:RNA, Neoplasm / Q000032:analysis / Q000235:genetics*; D011933:Reagent Kits, Diagnostic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016707:Tissue Fixation
D005404:Fixatives; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011933:Reagent Kits, Diagnostic; D005557:Formaldehyde