RNA extraction

felicity_austen at my-deja.com felicity_austen at my-deja.com
Tue Sep 7 05:29:54 EST 1999

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.

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