Fortunately I have all the RNA extracted for the next-gen project and so all I had to do was synthesize cDNA and amplify then sequence the genes of interest. I'll update with results.
So far 6 of the 7 candidates that have diagnostic fixed differences have been sequenced. The results remain the same as before: no breakdown of imprinting in one hybrid: there are some genes where breakdown occurs in both hybrids and some where imprinting is correctly maintained. Furthermore, there are no differences between the two sexes in the pattern of expression.
The 7th gene, Peg3, is not amplifying and so I can't sequence it. It is an important gene to assay because it is one of the main partners in the deer mice that cause parent-of-origin growth (Vrana et al, 2000). It is frustrating that it won't amplify reliably as samples I ran a year ago were great. The primers do not include an intron and so the first thing I will do is to try the primers on gDNA instead of cDNA - maybe the gene is not expressed highly in the placenta of hamsters and that is why the PCR won't work.
update 7-15-13: The PCR with gDNA worked fine, not a brilliantly blazing as the first time I did it, but not bad. This leaves me with the question of whether the original PCRs had gDNA contamination with the RNA. I think that this is not the case as Peg3 showed imprinted expression in those samples. If gDNA was a contaminate, the sequences would have been heterozygous and shown what I would have interpreted as biallelic expression. The only way imprinted expression could be found is if the gene is truly imprinted and there was no gDNA contamination.
Now the problem remains of how to get the Peg3 primers to work on cDNA like they did before. I have increased the extension time by 20 seconds in case the full 1000bps weren't being copied and I increased the cycle number from 35 to 40. Although I'm a little skeptical of amping anything 40 cycles, if there are faint bands there, 40 cycles should make them show up.
update 7-16-13: All the cDNA bands are really faint if they're there at all. I'm pretty sure the positive is present, but it's kinda shitty all the way around. E~ suggested that I should do a comparison between the gDNA of the original sequences and the gDNA of the one's I'm struggling with now to tell if there has been a problem with the primers (all gDNA's fail) or perhaps a SNP in the priming site (new gDNAs fail, old ones work). Although this is going against the Prime Directive (Science's General Order no. 1: only change one variable at a time) I am using new Taq too. This is partly because we are out of the old, and partly because I think the old may not be working. Here is my predictions/interpretation table:
IF: Then:
Both old and new gDNA fail primers are bad, reorder primers
Old gDNA works, new fails SNP in the priming site, redesign primers
Old gDNA fails, new works WTF, I have no idea what this would mean, I hope it doesn't happen...
Both old and new gDNA work old Taq was bad, the new is good, re-try with cDNA
update 7-30-13: here is the gel:
Land 1 is the ladder, lanes 2-7 are the samples, the first three are the old gDNA, the second three are the new gDNA, lanes 8 and 9 are the positive and negative control, lane 10 is another ladder.
All the gDNAs worked, so I will assume that the issue was bad Taq and tomorrow I will run out the cDNA with the good Taq.
update 8-1-13: Didn't work. The old cDNA still doesn't amplify. Try making new cDNA
update 8-14-13: I figured it out! I had cleaned the cDNA in the previous trials and since I had started with so little, I lost most of it. This time I did not clean the cDNA and have beautiful bands. Now for sequencing!
Lane 1 is the ladder, 2-18 are Peg3 from cDNA, 19 is the positive control and 20 is the negative control.
Such a ton of work for so simple a problem. I'm just glad it's working.
Vrana, P. B., J. A. Fossella, P. Matteson, T. del Rio, M. J. O'Neill, and S. M. Tilghman. 2000. Genetic and epigenetic incompatibilities underlie hybrid dysgenesis in Peromyscus. Nat Genet 25:120–124.
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