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In the reciprocal experiment, liver extracts were immunoprecipitated with antibodies against individual putative substrates, followed by detection of their acetylation abundance (reverse validation).
Results of forward and reverse validation showed that tubacin treatment resulted in elevated acetylation abundance of all three substrate candidates in 293T cells (Fig. 2D F).
MYH9 and DNAJA1 from HDAC6 knockout mouse liver showed increased acetylation signal when compared with those from the wild-type tissue (Fig. 2B), whereas acetylated Hsc70 was undetectable in the reverse validation experiment.
To compare our transcriptomic data with the knowledge derived from functional studies in the field, as a means of reverse validation, and in the absence in KEGG of pathways encompassing all enzymatic and signaling stress-related pathways, we directly assessed whether differentially expressed data, as detected by GSEA analysis in Abcd1 − mice, were enriched in genes involved in oxidative stress.
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The approach complements reverse genetic validation (transfection) of gene candidates which may be restricted to limited numbers of pre-defined genes and gene mutations (allelic replacements).
The average classification error in standard and reversed cross-validation scheme were very close to each other, in all cases the error is only 1percentnt higher in the reversed setup.
The forward and reverse primers for validation were listed in Table 1.
Interestingly, both microarray and reverse transcriptase PCR validation showed that BPA facilitated more significant down-regulation of ERβ than DHT.
For expression analysis we used next-generation RNA-sequencing (RNA-Seq) and for validation reverse transcription quantitative PCR (RT-qPCR).
What will validation validate?
Reverse-transcribed PCR validation of the C. cladosporioides MD2 unigenes.
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