We do some LOH as well as microsatellite instability. I have some pictures
on my website at www.hci.utah.edu/groups/genomics/services.htm which show
the expected results.
I have looked at microsatellite instability using the markers Roche has in
its kit, and although BAT25 and BAT26 are very sensitive for MIN, I don't
think they would be appropriate for LOH because of the 1bp up and down
ladder. The other primers should work fine.
A lot of the trouble we have with LOH or MIN is template dependent:
* Template prepared from archived tumor samples which have
who-knows-what kind of junk in them. Try primers with short product <200bp,
titrate the starting sample concentration to see how low you can go (to
dilute any PCR inhibitors), and use a hot start enzyme--we like Life Tech's
Taq Platinum.
* Tumor samples with a high proportion of normal cells. Get someone
really competent to microdissect the tumor slice so you get just tumor. For
some tumors (prostate for example) you're just going to have to live with
high normal concentration, so you have to do the experiment several times
and do the math to see if you are seeing a statistically significant
difference or not.
Hope this is helpful,
Linda
> ----------
> From: sharon.palmer@spectrum-health.org
> Sent: Wednesday, February 9, 2000 9:08 AM
> To: Recipients of ABRF List
> Subject: LOH
> Importance: High
>
> Hi all,
>
> I thought that someone in the group might be of some assistance. We are
> attempting to do a study concentrating on the loss of heterozygosity using
> microsatellite detection. We have tried the Roche kit and other specific
> labeled primers. The results are ambiguous at best.
>
> If any has a protocol, suggestion or has used the ABI rer/loh kit, please
> send a quick note.
>
> Thanks!!!!
>
> Sharon Keely Palmer
> Spectrum Health
> 616.356.4062
>
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