Loss of detectability of Charcot-Leyden crystal protein transcripts in blood cells after treatment with dimethyl sulfoxide.
Charcot-Leyden crystal protein (CLC) is a major secretory effector protein of eosinophils. In addition, CLC has been identified as marker for regulatory T-cells and differential expression of CLC has been described under diverse pathological conditions. By analysis of DNA microarray data from peripheral blood mononuclear cells (PBMC) we found differences for the expression of CLC between PBMC that had been cryopreserved or had been used for RNA isolation immediately after cell separation. Reverse transcriptase-polymerase chain reaction (RT-PCR) of separated cell populations indicated that contaminating granulocytes were the main source of CLC transcripts in PBMC. CLC was only detectable in fresh PBMC and not in cryopreserved material. Transcripts corresponding to CLC were also detectable by RT-PCR only in fresh PBMC and eosinophils. Loss of CLC transcripts in PBMC was induced by a short pulse with dimethyl sulfoxide (DMSO), indicating that the freezing medium was responsible for this phenomenon. We conclude that CLC transcripts are lost during cryopreservation in the presence of DMSO and can never be identified as differentially expressed in cryopreserved samples.
Foell
Juergen L
JL
Department of Pediatrics, Children's Cancer Research Centre, Martin-Luther-University Halle-Wittenberg, Halle, Germany.
Volkmer
Ines
I
Giersberg
Corinna
C
Kornhuber
Malte
M
Horneff
Gerd
G
Staege
Martin S
MS
D000943:Antigens, Differentiation / Q000096:biosynthesis*; D015925:Cryopreservation / Q000379:methods; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D004804:Eosinophils / Q000166:cytology / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation / Q000187:drug effects*; D006023:Glycoproteins / Q000096:biosynthesis*; D006098:Granulocytes / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008245:Lysophospholipase / Q000096:biosynthesis*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D012333:RNA, Messenger / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
D000943:Antigens, Differentiation; D003451:Cryoprotective Agents; D006023:Glycoproteins; D012333:RNA, Messenger; D008245:Lysophospholipase; C011711:lysolecithin acylhydrolase; D004121:Dimethyl Sulfoxide