Computational protocol: Sustained Spindle Assembly Checkpoint Response Requires De Novo Transcription and Translation of Cyclin B1

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Protocol publication

[…] Total RNA was isolated from NIH3T3 using Trizol® reagent (Invitrogen™) and cDNA synthesis was performed using M-MuLV reverse transcriptase (Fermentas) according to manufacturer's instructions. cDNA from NIH3T3 cells was amplified using primers for mouse sequences of Bub3 (sense: 5′-CAGACTCGCTGCATCCGA-3′; antisense: 5′-CGGCCTTCGATGGAGCT-3′), BubR1 (sense: 5′-CGGGACGCAG GCCTC-3′; antisense: 5′-TGGGAAGCACGCTGGG-3′), Cdc20 (sense: 5′-CGATTT GCACTCACTGCTTCA-3′; antisense: 5′-CAGCGCGCAACCGG-3′), Cyclin B1 (sense: 5′-TGTGTGAACCAGAGGTGGAA-3′; antisense: 5′- CGGGCTTGGAGAGG GATTAT-3′), Mad1 (sense: 5′-ACTAGCCGTGGCCTCTGCT-3′; antisense: 5′-CAT CCCCAGTAGCTTGCTCC-3′), Mad2 (sense: 5′-GGTAGTGTTCTCCGTTCGATCT AGT-3′; antisense: 5′-GCAGGGTGATGCCTTGCT-3′), Securin (sense: 5′-TGAATG AAGAGAGAGGGCTGG-3′; antisense: 5′-AAAGGGTGTCTTCAGAGGGCTA-3′) and L19 (sense: 5′-GGAAAAAGAAGGTCTGGTTGGA-3′; antisense: 5′-TGATCTG CTGACGGGAGTTG-3′). L19 expression served as internal control and was used to normalize for variances in input cDNA. Primers were designed using the ABI Primer Express software and their specificity confirmed using NCBI BLAST module. Detection of the expression of each gene was performed with SYBR Green (Applied Biosystems) in an ABI PRISM 7700 Sequence Detection System (Applied Biosystems), using the relative standard curve method. All measurements were performed in triplicate. […]

Pipeline specifications

Software tools Primer Express, BLASTN
Application qPCR
Diseases Heart Arrest
Chemicals Nocodazole