cDNA

[Thomas Newman]

In article <D0IH4L.GsL at osuunx.ucc.okstate.edu>, Wenjun Huang
<biocwjh at osuvm1.bitnet> wrote:

> 	I need a help! (for my first strand cDNA synthesis from cotton cell
> suspension culture)
> 	The total RNA was isolated by use of extraction buffer (containing Tris,
> NaCl, EDTA and ATA), phenol/chloroform, LiCl precipitation,
> phenol/chloroform, ethanol/NHAc precipitation.  The ratio of A260/280 of
> the RNA is 1.9-2.0, the RNA yield is about 600 mg/10 g cells.
> 	The poly(A)+RNA was selected by oligo(dT) cellulose column (GIBCO BRL,
> ultra PURETM).  The ratio of A260/280  of the selected poly(A)+RNA is
> 1.98-2.02.  The RNA and poly(A)+RNA looked fine based on formaldehyde gel
> electrophoresis and in vitro translation.  
> 	I used ZAP-cDNA synthesis kit (Stratagene) to construct the cotton cDNA
> library.  However, I did not get the first strand cDNA.  Strangely, when
> I continued the second strand cDNA synthesis, I got smear band with
> reasonable sizes on developed X-ray film.  The test control RNA provided
> by the kit always worked.  Did my sample poly(A)+RNA degrade during cDNA
> synthesis? I added reaction buffer into sample poly(A)+RNA and incubated,
> then ran formaldehyde gel, the poly(A)+RNA was still there with a little
> bit degradation.   

 My understanding is that ATA directly modifies RNA and is fine for
northerns, but makes the RNA unsuitable as a template for Reverse
transcriptase.  I assume that is why your first strand synthesis is not
working.  I suggest a different RNA extraction such as a guanidinium based
buffer.

Tom Newman
MSU-DOE Plant Research Lab