4 replies to this topic

[proteinz]

hi guys! i am having a hair pulling moment here i cant determine what my northern blot results mean i hope someone can help...
i ran c. elegans RNA and detected with cDNA probe.  the length of the (cdna) gene is 594bp.  my gDNA sequence is approx 900bp .  i am getting two transcripts at approx 900bp and 1200bp what does this mean?
thanks in advance

Edited by proteinz, 01 August 2009 - 03:29 AM.

[eldon]

proteinz, on Aug 1 2009, 03:28 AM, said:

hi guys! i am having a hair pulling moment here i cant determine what my northern blot results mean i hope someone can help...
i ran c. elegans RNA and detected with cDNA probe.  the length of the (cdna) gene is 594bp.  my gDNA sequence is approx 900bp .  i am getting two transcripts at approx 900bp and 1200bp what does this mean?
thanks in advance

what are their relative intensities to one another?

how specific is your probe? do you get 2 signals on a Southern?

both could be your gene....size determination on denaturing gels is approximate, however, since the cDNA is predicted to be ~ 600bp you cannot be sure about the modifications your transcript might be subjected to.
the sizes could be due to variable length poly-A tail, variable 5' upstream elements, closely related gene, multiple copy gene, non-specific target, poor probe design, overloaded total RNA causing the ribosomal bands to appear on your film/phosphorimager etc.

[proteinz]

thanks for the reply eldon i'm trying to get this sorted for my thesis and im more of a protein girl as my name would suggest!!! your my only hope!

it seems to be some sort of highly conserved gene family. when i run it on a southern i get multiple i.e 4 bands. the lower 900bp band is more intense than the other. i was previously aware that there were two genes which were pretty much identical except for their intron region so the presence of two doesnt bother me its the size more so..

had thought about them being the subunits but they are appearing at a slightly higher size. would poly a tail and other upstream UTR adding an extra ~400-500bp be a normal occurance? when i did the blot in a similar nematode to the the same gene (different probe) i also got two bands but the sizes were what i had expected from the cDNA. would this discount the post translational modification in the other nematode or could this happen in one and not the other
thanks again!

[eldon]

mRNAs can be quite different in size and a polyA tail can be the reason, or it could be multiple modifications to the transcript.

different transcript sizes across species is possible...especially if the gene resides on different chromosomes btwn organisms...multiple copy genes that are identical in terms of the coding region, but reside on separate chromosomes in the organism may often have different sized mRNAs as a means of regulating gene expression or mRNA stability in different cell types.

can you enrich for cell types with your worm? this might aid in delineating differential transcript expression in different cell types.

is the probe different due to sequence variation? maybe try the same probe on each probe.

[proteinz]

many thanks again eldon your suggestions are very helpful.yes il try again with the same probe and see if that shows similar results.
hope you have a nice day!