RNase protection/PCR product

[Martin Leach]

In article <9312131730.AA29735 at post.its.mcw.edu>, khopp at POST.ITS.MCW.EDU
(Kathleen Hopp) wrote:

> Dear Bionetters,
>      I am attempting to implementt RNase protection to verify expression
> of a PCR product obtained by RT-PCR of material from cell culture. My
> protection probe (even after gel purification) always has multipke (maybe
> multitudinous is a better word) smaller bands. I've tried a couple changes
> in procedure but am ready to go all the why back to making new DEPC water.
>     Anyone have any advice or experience to offer? Altermatively is there
> another way to check if my PCR product is truly a reflection of a species
> present in the culture material? We have sequenced it and it reflects a 
> "double (read that  double) exon product where a alternative splicing
> situation is usually found.
> 
>     Please excuse the noise... it is not expletives!
> 
>    Thank you in advance,


Dear kathy,

Several things.

We have found that the digestion buffer has to be made fresh each time in
order to obtain single bands. We are not sure why, but when we do this the
digestion works better and leaves a lot less bands. Try experimenting with
using rnaseT1 alone. We use this alone for some things and it gives a
better banding pattern than using rnaseA alone or combined. However, rnaseA
is required for some reactions otherwise we don't get complete digestion.

An alternative method for checking this species:

1. do rnase treatments of the rna before rt-pcr, if it is rna it will not
pcr.

2. also do DNAse controls just in case the product is from DNA
contamination.

3. do a northern blot to check for the rna species, a lot simpler than
rnase protection.

4. you could perform s1 nuclease protection analysis (rna:dna hybrids) to
identify the species (although rnase protection is more conventional)

Good Luck


Martin Leach
-- 

.....          Martin Leach                Email:leach at mbcrr.harvard.edu 
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