[proteomics] Freeze Thaw issues with oligonucleotides
- From: Derek Donohue <shesgruesome@xxxxxxxxx>
- To: "proteomics@xxxxxxxxxxxxx" <proteomics@xxxxxxxxxxxxx>
- Date: Thu, 27 Oct 2011 14:08:58 -0700 (PDT)
Hi all. We recently saw some odd Cts from a qPCR based test we use for QC'ing
oligonucleotide plates. We ran tests and checked records on all of our
materials and found them to be in spec. However we found our (previously
in-spec) oligonucleotides to be out of spec for concentration. We assumed the
cause to be within tube heterogeneity that was aggravated by aliquotting off
the top layer. After broad investigation were ultimately led back to our
freeze thaw prodcedures.
As a check we thoroughly mixed the remaining oligos then separated them into
two aliquots. We then re-ran 2 new sets of plates after thawing one set of
oligos using our existing thawing protocol as well, and the other on a Box
Scientific thaw station (you set plates or tubes on it and it circulates
ambient air to give fast/even thawing). Interestingly enough we saw more
consistent results after using the thaw station. Cts were expectedly higher
but relative Cts gene to gene looked more in line with historical data. We
interpreted this as lower overall variation.
Does anyone have experience with identified issues in thawing proteins and
oligonucleotides that led to inconsistent or unexpected data?. In the course
of our investigation we were surprised to find lots of papers on the topic but
didn't feel many were relevant to our exact model. But now that we have proven
the point to ourselves, we are considering a wider study in house. There is
little regulatory guidance around thawing itself, so we are just curious what
insights the greater community may be able to offer. We are also curious if
anyone has any broader experience with these thaw stations? We bought one out
of curiosity but haven't used it much. However it clearly gave better results
in our study, and we see now that it allows us to determine an optimum thaw
time then proceduralize (is that a word? MS says no) it. Seems like better
practice anyway.
Anyway, just looking for thoughts on all topics. I'm new to the forum so hello
and thanks for your comments.
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- » [proteomics] Freeze Thaw issues with oligonucleotides - Derek Donohue
