different transcription from 5' UTR than 3' UTR primers - (May/24/2008 )
Hello everyone,
I have a gene that I am trying to understand it's transcription. Briefly, it has two different splice products, one long and one short. When I orignally started in on this project, they had a primer for the long and the total. THe total was from the 5' UTR. The first problem is that the long product is more transcribed than the total (from qrt-pcr). So I designed primers for the short (exon spanning) and more primers in the 5'utr and 3' utr. One more complication, when the RNA is taken from a cancerous cell line, there is more long than short. Now, my new primers in the 5' utr region acts the same as the previous total primers. But, in the new 3' utr primer, there is more transcribed in only the cancerous cell rna. Does anyone have any idea what this could mean. I was anticipating if anything, more transcription from the 5' end, since this is where transcription initiates?? 
I have a gene that I am trying to understand it's transcription. Briefly, it has two different splice products, one long and one short. When I orignally started in on this project, they had a primer for the long and the total. THe total was from the 5' UTR. The first problem is that the long product is more transcribed than the total (from qrt-pcr). So I designed primers for the short (exon spanning) and more primers in the 5'utr and 3' utr. One more complication, when the RNA is taken from a cancerous cell line, there is more long than short. Now, my new primers in the 5' utr region acts the same as the previous total primers. But, in the new 3' utr primer, there is more transcribed in only the cancerous cell rna. Does anyone have any idea what this could mean. I was anticipating if anything, more transcription from the 5' end, since this is where transcription initiates??
I don't understand, what do you mean by transcription from primers?
I probably using the wrong word. After analyzing the ct values versus actin, the 3' utr shows higher transcription/expression....
I have a gene that I am trying to understand it's transcription. Briefly, it has two different splice products, one long and one short. When I orignally started in on this project, they had a primer for the long and the total. THe total was from the 5' UTR. The first problem is that the long product is more transcribed than the total (from qrt-pcr). So I designed primers for the short (exon spanning) and more primers in the 5'utr and 3' utr. One more complication, when the RNA is taken from a cancerous cell line, there is more long than short. Now, my new primers in the 5' utr region acts the same as the previous total primers. But, in the new 3' utr primer, there is more transcribed in only the cancerous cell rna. Does anyone have any idea what this could mean. I was anticipating if anything, more transcription from the 5' end, since this is where transcription initiates??
I don't understand, what do you mean by transcription from primers?
I am still not sure I exactly understand the problem, so I will just say a few things if that helps.
1. When you do RT using Oligodt priming, the 3' side of RNA is more represented than the 5' side of the RNA, because the RT starts at 3' end and may fall off before reaching the 5' side.
2. 3' and 5' UTR areas somtimes have secondary structure difficult to negoitate for the RT, so if your 5' UTR secondary strcuture was not resolved at the usual RT reaction temp of 42'C, you need to use RT that allows upto 65'C.
hmmm, I guess that could be some of the issue, I do use oligo T priming. It's weird though since my "normal" cells don't have enhanced expression at the 3' end, only the cancerous cells. So you would think that oligo dt priming would effect all cell lines universally?? My cDNA temp is at 55 degrees already (superscript III by invitroge). As far as secondary structure, I don't think that is the issue. I plugged the structure into a program that analyzes that, I think it's oligos website, by giving you -deltaG values as well as use primer3 to design the primers.
I am still not sure I exactly understand the problem, so I will just say a few things if that helps.
1. When you do RT using Oligodt priming, the 3' side of RNA is more represented than the 5' side of the RNA, because the RT starts at 3' end and may fall off before reaching the 5' side.
2. 3' and 5' UTR areas somtimes have secondary structure difficult to negoitate for the RT, so if your 5' UTR secondary strcuture was not resolved at the usual RT reaction temp of 42'C, you need to use RT that allows upto 65'C.
That may mean your cancerous cells RNA is (more) degraded, hence higher representation of 3' ends when using oligodT. You may want to use random priming in that case.
..
why would that be, I give all the cells harvested the same treatment?
That may mean your cancerous cells RNA is (more) degraded, hence higher representation of 3' ends when using oligodT. You may want to use random priming in that case.
..
1. More number of cells when you harvest, even if you have seeded same number of cells - This is possible, because cancer cells may grow faster, wouldn't care about monolayer business..
2. Your particular gene may be expressing supremely, and so the relative number of 3' ends may be more.
3. There is a difference in this gene's sequence in normal vs. cancer cells, leading to a differnce in RNA turnover.
2. Ultimately, you treat all cells same, but remember, these are different cells, so nature may be treating them differently (i.e. cells may have faster degradation machinery, higher metabolism and subsequent lack of nutrition, higher overall RNA turnover, higher number of dead cells with degraded RNA, and God knows what else).
As such, while I always think of wildly fanciful reasons, I always favor simple explanations. And the simplest one is that there is something technically different in the way you treat this cells vs. normal cells.
Thanks a bunch by the way for talking this out with me, it's fun to talk to someone else and work it through. I am pretty sure the transcription rate is higher in my gene in the cancer cells, in fact, we are close to submitting a paper to Blood on that data. We were actually using qPCR using hnRNA versus mRNA to make sure that degradation of the message is not part of the mechanism (an additional technique done along with actinomycin D), along with discounting alternative splicing and other post transcriptional mechanisms of regulation. Ultimately, for this type of analysis it is important which mRNA I use, that detected with the 5' UTR or that detected with the 3' UTR. Oh, by the way, I always use 2 x 10^6 cells for RNA extraction, and 1 ug of RNA for cDNA synthesis.
1. More number of cells when you harvest, even if you have seeded same number of cells - This is possible, because cancer cells may grow faster, wouldn't care about monolayer business..
2. Your particular gene may be expressing supremely, and so the relative number of 3' ends may be more.
3. There is a difference in this gene's sequence in normal vs. cancer cells, leading to a differnce in RNA turnover.
2. Ultimately, you treat all cells same, but remember, these are different cells, so nature may be treating them differently (i.e. cells may have faster degradation machinery, higher metabolism and subsequent lack of nutrition, higher overall RNA turnover, higher number of dead cells with degraded RNA, and God knows what else).
As such, while I always think of wildly fanciful reasons, I always favor simple explanations. And the simplest one is that there is something technically different in the way you treat this cells vs. normal cells.