Quantitation of non-polyA mRNA - (Jun/11/2008 )
I'm exploring relative gene expression in two different chloroplast-deficient mutants strains of Arabidopsis. I'm working in a small lab at a small college, so we just received our first ever qPCR machine, which was very poorly documented and I've basically had to figure out myself, so a lot of this stuff is new to me. I've had good success doing qPCR on nuclear encoded genes by using cDNA reverse transcribed using OligoDT and total RNA. However, most chloroplast-encoded genes aren't polyadenylated, so this won't work for determining expression of chloroplast-encoded genes. If I were to reverse transcribe cDNA using reverse primer for a gene of interest along with reverse primer for a normalizer, would this at all reflect relative expression of the gene? Wouldn't differences in primer efficiency change the rate of reverse transcription and thus the ratio of chloroplast-encoded gene to normalizer would change through the cDNA synthesis? Is there any way around this, short of doing a Northern blot (we are a small lab without a radiation cert.)? Also, does anyone know of a program out there that can compare multiple primers for primer-primer interactions? I was hoping to perform just one reverse transcription using a primer cocktail for several genes at once to conserve RNA (our mutants are tiny and hard to grow so RNA is at a premium), but while everything I've found tells me to watch out for primer-primer interaction, it doesn't tell me just how I might check for that.
i don't understand the problem. why can't you just use random primers instead of oligodT primers for reverse transcription?
That would be my answer right there. Thanks. Sorry if it seemed like a rather simple question, I'm kind of figuring this stuff out on my own with little support from my supervisors and only a bachelors degree under my belt. There is a lot of information out there that is hard to find simply because people think of it as being a no-brainer.
As you already got the answer, just a small comment - Northern blot can be done using non-radioactive labelling, but - anyhow - qPCR has a number of advantages so I would stick to that.