css at med.unc.edu
Wed Sep 8 16:33:46 EST 1999
Perhaps a mini-RNA kit that utilizes spin columns would be useful. I
don't think you're out of range with 0.1 g starting tissue. Micro Fast
Track (Invitrogen, maybe?) is one kit that comes to mind. I'm sure
there are others.
felicity_austen at my-deja.com wrote:
> I am an honours student trying to get RNA for cDNA subtraction (Clontech
> Cells are PHA cultured, and non-cultured rainbow trout thymocytes.
> Lymphocyte collection is centrifugation on density gradient (Ficoll).
> RNA extraction with residual Ficoll contamination doesn't work.
> Hypothesise that Ficoll binds RNA and takes it to organic phase of
> extraction. Ficoll does not inhibit cell lysis by my determination.
> Any leads to references regarding effects of Ficoll on RNA extraction
> greatly appreciated.
> Starting material extremely limited. Tissue mass 0.1g. Lymphocytes
> collected from tissue. Cells from different fish not pooled to avoid
> mixed lymphocyte reaction. Cell culture decreases cell numbers. Only
> fish whose thymocytes proliferate in PHA culture (ignoring cell death
> (determinated by BrdU (thymidine analog) incorporation assay)) are used
> for RNA (1/3 to 2/5 of fish respond well for me).
> Limitation of starting material rules out RNA binding kits currently
> available to me for extraction (eg Qiagen) as retained RNA will probably
> equal my entire sample. I use acid guanidium phenol chloroform method
> similar to Chomczynski (1986?). A lot of genomic DNA is in my samples.
> Intended use of RNA is cDNA subtraction which means I cannot have
> genomic DNA (as little as technically possible). Try RNase-free DNase
> digestion, but lost one of three samples (RNase present in single
> sample?). Try initial ethanol precipitation after lysis (1/3 vol Abs
> EtOH) to remove DNA contamination (ref: possibly Chenchik). I removed
> DNA, I also had no RNA. Suggested I did not achieve critical mass of
> RNA to start precipitation. Try DNase digestion with RNase inhibitor
> but now both reactions ate RNA. DNase enzyme and RNase inhibitor (from
> Promega) were first opened for this reaction. Sample had been shown to
> survive reaction temperatures and times without addition of DNase.
> Pipettes and filter tips were clean. Only possibility I can think of
> for RNase contamination is tubes (DEPC treated, but opened previously).
> Any ideas on how to fix DNase digestion or how to avoid DNA in first
> instance would be monumentously appreciated.
> Sent via Deja.com http://www.deja.com/
> Share what you know. Learn what you don't.
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