dspinella at chugaibio.com
Wed Sep 30 09:10:30 EST 1998
> Hi Netter
> I am trying Reverse Northern to analysis the cDNA fragments obtained
> from DD-RT-PCR. But after autoradiography, My film is clear. I think
> hybridization process have some problem. I don't know why no dot is
> I blotted PCR product on zeta-probe membrane with Bio-Rad Dot apparatus.
> I denatured the 30ul PCR product, added NaOH and EDTA, and heated it.
> I neutralized cDNA samples with Tris buffer and loaded it to Dot
> I am using Rapid-Hyb buffer manufactured by Amersham as Hybridiztion
> Hybridization was done at 65 C for 3 hours
> Membrane washing was done at 65 C for 15 minutes two times with 0.7X
> cDNA probe was produced from MMLV reverse transcriptase-using reaction
> with 20 ug RNA. I think its CPM is approximately 40 CPM.(Recently I
> didn't count CPM, because
> I think the step is relatively simple.
> Is there any problems in Blot or Hybridization processing?
> Is there other recipe for Zeta probe membrane or for Reverse Northern
> Could you advice me to solve my troubles?
> Kookkyung lee
> Korea Food & Drug Administration
> Department of Pathology
First of all if, your total cDNA probe has truly only 40 CPM of
incorporated isotope, something is seriously wrong (this is less than
background level). I'll assume that this is a typo and we're talking
more like 40,000 CPM. The more likely problem is that the specific
message in the total cDNA probe is at too low a concentration to
hybridize to the solid phase target in any reasonable length of time.
Many low abundance messages fall into this category and are difficult to
make work in reverse Northerns. Are you using 20 ug of total RNA or
poly A+ RNA to make your probe? If you have started with total RNA, try
using the same amount of poly A+ RNA to effectively increase the
concentration of the specific message you probing for. Also, anything
you can do to decrease the volume of the hybridization will also help.
You may have to just bite the bullet and do a standard Northern using
your PCR product as probe and elctrophoresed and blotted PolyA+ RNA as
your solid phase target. Good luck. --Dom Spinella
More information about the Methods