From bambo_hn yahoo.com Tue Jan 31 08:35:04 2012
From: Minh Nguyen <bambo_hnyahoo.com>
Subject: Re: How to Extract Leishmania DNA from blood of an infected
mouse?
To: Multiple Recipients of <angenmapanimalgenome.org>
Date: Tue, 31 Jan 2012 08:35:04 -0600
Hi Joseph,
I have no direct experience on Leishmania DNA extraction from blood.
However, I would like to share with you some of my thoughts. Generally, in
order to obtain good PCR products, a number of factors involved: enough
quantity and good quality of gDNA, a good PCR protocol, a good design
of a primer pair and good quality DNA polymerase. In your case, all factors
may be considered. However, not sufficient amount of gDNA could be the main
cause. I remember, but not sure 100 %, that the density (concentration) of
Leishmania is less in blood than in spleen and liver and it could be peak or
flat depending on the cycle (in a day or a period of days). If it is true, you
can try to collect blood samples at the moment when Leishmania multiplies at
high level. Second thing you could do is to use “home made” Proteinase K
digestion, Phenol-Chloroform extraction, and ethanol precipitation for gDNA
extraction. This traditional protocol is the best to recover most of gDNA with
reliable quality. With kits and column, gDNA may be lost especially when you
start with little material. Third thing, just assuming that there are a few
Leishmania cells in a volume of blood and what you could try is to start with
more (10 x) volumes of blood for example. If there is no Leishmania cell in
blood, certainly nothing could work.
If you need any information, please send me an email.
Hope this will help,
Minh Nguyen
________________________________
From: Joseph Kamau
.To: Multiple Recipients of
.Sent: Tuesday, January 31, 2012 9:27 AM
.Subject: How to Extract Leishmania DNA from blood of an infected mouse?
Dear all,
I am looking for a DNA extraction method that can be used to extract
Leishmania parasite DNA from blood of an infected mouse. I have tried using
Quick-gDNA MiniPrep, but the DNA concentration is too low to be detected by
even LAMP or Nested PCR.
When the kit is applied on liver and spleen tissues of the same infected
mouse, the amount of parasite DNA extracted is quite much, but tissue
biopsies cant be the routine diagnostic method of choice.
Blood would be the better option if we would find a way of extracting
sufficient parasite DNA.
Some people have recommended going for RNA as opposed to DNA, that parasite
RNA would be much! am yet to understand the rationale.....
kindly anyone out there who can help navigate the problem?
Sincerely,
Joseph Kamau, PhD
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