RNA extraction

Caroline Szymeczek-Seay 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.

caroline

felicity_austen at my-deja.com wrote:
> 
> I am an honours student trying to get RNA for cDNA subtraction (Clontech
> Kit).
> 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.
-------------- next part --------------
A non-text attachment was scrubbed...
Name: css.vcf
Type: text/x-vcard
Size: 146 bytes
Desc: Card for Caroline Szymeczek-Seay
Url : http://iubio.bio.indiana.edu/bionet/mm/methods/attachments/19990908/fc5f2778/css.bin


More information about the Methods mailing list