Field of Science

Next steps with the old array data

What do I do with the array Background values?  I was thinking I should divide by them, but I looked online and found that the usual practice is to subtract each spot's Background value from its Signal value.

I also need to figure out how to properly compare the effect of the purR knockout on purine genes to the(lack of) effect in the sBHI time course arrays.  All the slides in the time course used the same 'reference' RNA (a pool of all the time point RNAs), but the purR analysis used RNA from purR+  cells in mid-log + cAMP.

Once I've figured this out I can do the next step with the array data:  I'd like to compare rec2's amount of induction in late log (relative to other CRP-S genes) to the difference in its relative expression caused by the purR knockout.  Maybe I'd better try saying this another way:  I want to get expression ratio data for rec2 and some other CRP-S genes, both from the time course arrays and from the purR+/purR- array.  In late log in sBHI (the time course), the CRP-S promoters are a bit more active than in early log, but PurR is keeping the purine genes off.  In the purR+/purR- microarray, the expression of the CRP-S genes is probably comparable to the time course, but the purine genes are on full strength.  If PurR doesn't normally repress rec2, the ratio of rec2 RNA to the other CRP-S RNAs should be the same in the time course arrays and the purR+/purR- array.  If PurR does normally repress rec2, the ratio should be higher in the purR+/purR- array.

I don't want to go to the trouble of extracting the data for all the CRP-S genes, so I'll just do rec2, HI1182/1183, and dprA.  No,,wait, the post-doc says he can easily write an R script to do this, so I'll wait until he's finished writing his NIH fellowship application.  In the meantime I'll fix all the flaws in my purine gene analysis.

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