4 replies to this topic

[Andras]

My brain hurts, so I ask for help.

This is the first time I'm doing Northerns, and my task is simple: I need to describe the dynamics of Sindbis virus replication. That means detecting the amount of positive and negative strand RNAs, and, if possible, the amount of the subgenomic RNA...

But I do have questions...
First, the oligo design looks relatively simple: just a 40 or so nucleotide long complementer sequence to the positive or negative strand -right?
How do I validate them? (To show that they indeed are specific.)

How do I show the different types of RNAs, and their ratios? I was thinking of a probe that spans both the subgenomic RNA and the complete RNA, hence it would detect all three forms: complete, and the subgenomic. Is that a valid approach?

Some people use RNA probes instead of DNA ones - how should I choose one or the other? The problem is that I need to do it quick and dirty - I have to characterize my system, so I can actually start "THE" experiment, which will take months or more to perform and evaluate, so I don't have many shots at it. My funding runs out in 18 months. (No pressure, of course.)

It was suggested that I dig up oligos by people who used them before, but after a day reading paper after paper I still have not been able to find probe sequences -if someone happen to know them, I'd be greatful.

Thank you for your time.

Edited by Andras, 05 April 2011 - 09:09 AM.

[bob1]

Is the Sindbis genome sequenced  and published on NCBI(at least partially)?  If so, you can use that sequence for probe design and BLAST the probe sequences to see if they will bind to anything else, especially the genome of the cells you are growing the virus in.  If not, I presume you have enough sequence to design probes, which can then be BLASTed as above.

I don't think you will be able to use a single probe to detect the whole genome -that would be a very large probe. Trying to probe more than one type of RNA at a time will probably get you into trouble as how will you differentiate which is which?

RNA:RNA is more stable than RNA:DNA, so the signal lasts longer and is less prone to degradation by pH etc., however, it is really really easy to generate lots of DNA and label it strongly (e.g. PCR labelling), whereas RNA labelling is a bit more tricky and tends to be only labelled at one end.  I would talk it over with your supervisor.  Have a look at a good manual, such as the ROCHE DIG applications for filter hybridisation manual, which if you ignore all the stuff selling your DIG, has lots of useful information and troubleshooting.

Good luck... they can be tricky.

[Andras]

bob1, on 05 April 2011 - 03:55 PM, said:

Is the Sindbis genome sequenced  and published on NCBI(at least partially)?  If so, you can use that sequence for probe design and BLAST the probe sequences to see if they will bind to anything else, especially the genome of the cells you are growing the virus in.  If not, I presume you have enough sequence to design probes, which can then be BLASTed as above.

I don't think you will be able to use a single probe to detect the whole genome -that would be a very large probe. Trying to probe more than one type of RNA at a time will probably get you into trouble as how will you differentiate which is which?

RNA:RNA is more stable than RNA:DNA, so the signal lasts longer and is less prone to degradation by pH etc., however, it is really really easy to generate lots of DNA and label it strongly (e.g. PCR labelling), whereas RNA labelling is a bit more tricky and tends to be only labelled at one end.  I would talk it over with your supervisor.  Have a look at a good manual, such as the ROCHE DIG applications for filter hybridisation manual, which if you ignore all the stuff selling your DIG, has lots of useful information and troubleshooting.

Good luck... they can be tricky.

Thank you for the quick answer. I have the probes designed, and BLAST shows nothing else coming up -can I presume that they're specific? (I don't know what target-validation procedures are used in Northern. If it was a PCR, I would just send the product to sequencing.)
The genome is a single straded RNA: no matter what probe I use, it'd show it at the exact same spot. The subgenomic RNA is also a separate molecule at a distinct size - so if everything's perfect (right...) I should get two bands using an overlapping probe. At least this is what I think...
Thank you again. I'll give it a try.

[bob1]

Ah, yeah, forgot about the ssRNA thing.  It should be fine, though I have never done northerns on an RNA based organism.  Is the subgenomic RNA a single strand as well, and is it complementary to the genome?

[Andras]

bob1, on 06 April 2011 - 07:02 PM, said:

Ah, yeah, forgot about the ssRNA thing.  It should be fine, though I have never done northerns on an RNA based organism.  Is the subgenomic RNA a single strand as well, and is it complementary to the genome?

Thank you for the answer. Yes, the subgenomic is a single-strand RNA as well, transcribed from a full length negative strand genome.
I'll see next week, hopefully...