about RNA expression analysis

[ho&ho]

anyone can shed some light on these puzzles as bellow, thanks.

 

Microarrayanalysis has identified 3 previously uncharacterised genes that are strongly up-regulated in embryonic stem cells exposed to retinoic acid (embryonic morphogen).

 

what assay can be applied to identify :

1. How many splice forms of each gene?

2. How big are the splice forms?

3. Where do each of the mRNA’s produced start and finish

4. Are they expressed in the developing embryo?

5. Where are they expressed?

6. Are they expressed in adult tissues and if so where?

 

i think

1. northern blotting/ AgaroseGel Electrophoresis /

2.

3. In situ hybridisation, but which one would be better, Sectional analysis or Whole mount analysis?

4.

5.

6.

[Bluenoise]

1. How many splice forms of each gene?

2. How big are the splice forms?

3. Where do each of the mRNA’s produced start and finish

4. Are they expressed in the developing embryo?

5. Where are they expressed?

6. Are they expressed in adult tissues and if so where?

 

Though this is homework, I'll resist my regular intentions and answer anyways.

 

Your answer for #1 is viable and will also provide a rough estimate for #2.

Sequencing of the fragments obtained by your anwer for #1 will provide a definate answers for 2; and 3 when compared to genome sequence.

Microarrays can be done for 4 and first part of 6.

In-situ can be done for 5 and the second part of 6.

 

There are also other possibilites, but the ones I've listed are probably the easiest / most practical.

 

Edit to tell you the truth 4+5 would be done easiest using a reporter gene assay however this wouldn't tell you which spiced form is present, only that one is. However as the question doesn't specify this either way you may want to say that.

[ho&ho]

thx a lot.

 

i m much clear at #4,5,6 by ur post ,but still wondering how to sort #3 via comparing to genome sequence?

 

Where do each of the mRNA’s produced start and finish?.

 

your anwer for #1 will provide a definate answers for 2; and 3 when compared to genome sequence.

.

 

i think the #3 is about the location where the mRNA is produced, ist it?

[Bluenoise]

No #3 is about the location of the mRNA in the genome, and the start and stop of the exons that are in the genome.

[ho&ho]

got it , that is why to compare with genome sequence... thx u very much indeed.

[CharonY]

But mRNAs do not start with the exons, wouldn't a RACE PCR for instance be more viable for #3?

[Bluenoise]

Well no they don't start with exons. But sequencing the mRNA will give you both the start/stop of the mRNA and each individual exon for the included spliced forms.

 

Anyways RACE PCR is just a very specific method for sequencing the ends. Whose terminology most definatley falls out of the range of the given assingment

[CharonY]

Well, it depends. If I refer to mRNA I usually mean the complete transcribed molecule. That is, including the upstream non-coding regions (including promotors and RBS). But this might only be a matter of terminology.

[Bluenoise]

Well, it depends. If I refer to mRNA I usually mean the complete transcribed molecule. That is, including the upstream non-coding regions (including promotors and RBS). But this might only be a matter of terminology.

 

No you're quite right, and that's what I've been saying all along.

[CharonY]

Upon rereading I found that I misunderstood you. I assume I was thinking of ESTs for some reasons (or for any other reasons I assumed you meant to sequence exons or whatever).

Of course RACE or other essays essentially only lead to same that you propose:

getting the sequence of the mRNA.

My apologies for the distraction.