An improved strategy and a useful housekeeping gene for RNA analysis from formalin-fixed, paraffin-embedded tissues by PCR.
Specific amplification of nucleic acid sequences by PCR has been extensively used for the detection of gene rearrangements and gene expression. Although successful amplification of DNA sequences has been carried out with DNA prepared from formalin-fixed, paraffin-embedded (FFPE) tissues, there are only a few reports regarding RNA analysis in this kind of material. We describe a procedure for RNA extraction from different types of FFPE tissues, involving digestion with proteinase K followed by guanidinium-thiocyanate acid phenol extraction and DNase I digestion. These RNA preparations are suitable for PCR analysis of mRNA and even of intronless genes. Furthermore, the universally expressed porphobilinogen deaminase mRNA proved to be useful as a positive control because of the lack of pseudogenes.
Finke
J
J
Medizinische Klinik, University of Freiburg, FRG.
Fritzen
R
R
Ternes
P
P
Lange
W
W
Dölken
G
G
D001483:Base Sequence; D002478:Cells, Cultured; D003584:Cytological Techniques; D004247:DNA / Q000235:genetics; D005557:Formaldehyde; D006801:Humans; D008969:Molecular Sequence Data; D010232:Paraffin; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000235:genetics* / Q000302:isolation & purification; D017423:Sequence Analysis, RNA / Q000379:methods
D005557:Formaldehyde; D012313:RNA; D010232:Paraffin; D004247:DNA