IDH data was obtained for a subset of the samples based on availability. IDH mutations were determined as part of a separate project, as described elsewhere [20 (link), 21 (link)]. Briefly, for IDH1 and IDH2 sequencing, genomic DNA was isolated from formalin-fixed paraffin-embedded or frozen tissue using the Recoverall Total Nucleic Acid Isolation Kit (Invitrogen, Grand Island, NY). Sequences of IDH1 at residue 132 (R132 for wild type, CGT) and IDH2 at residue 172 (R172 for wild type, AGG) were determined by Sanger sequencing with the following primers: IDH1 (forward, 5′-gcgtcaaatgtgccactatc-3′ and reverse, 5′-gcaaaatcacattattgc-caac-3′) and IDH2 (forward, 5′-CTCACAGAGTTCAAGC TGAAG-3′ and reverse, 5′-CTGTGGCCTTGTACTGCA GAG-3′). Purified PCR products were sequenced using BigDye Terminator v1.1 and analyzed on a 3730 sequencer (both Applied Biosystems). For some samples sequences around codon 140 of IDH2 were also obtained (R140 for wild type, CGG).
Protocol detail
Targeted IDH Mutation Sequencing
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Corresponding Organization : The Lundquist Institute
Other organizations : UCLA Health, University of California, Los Angeles
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Variable analysis
independent variables
- IDH1 residue 132 mutation (R132 for wild type)
- IDH2 residue 172 mutation (R172 for wild type)
- IDH2 residue 140 mutation (R140 for wild type)
- Not explicitly mentioned
- Formalin-fixed paraffin-embedded or frozen tissue samples
- Primers used for IDH1 and IDH2 sequencing
- Sanger sequencing method
- BigDye Terminator v1.1 and 3730 sequencer used for sequencing analysis
- Positive control: Wild-type IDH1 (R132) and IDH2 (R172, R140) sequences
- Negative control: Not mentioned
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