Quantifying mRNA levels

[Sr_08]

Hi,

I am new to this field. Have not had much experience with RNA work.
Can anyone tell me how does one quantify mRNA ? I am doing induction studies and have to check the mRNA levels and how they differ at differnet induction levels/times
Thank you

[luisillo]

Real-time PCR (qPCR). The technique is similar to a conventional RT-PCR: You isolate your RNA, retrotranscribe it to cDNA and do the PCR. In qPCR case, you need special cyclers which can read the signal of dyes known as reporters. These dyes bind to new DNA strands, so, the higher the signal, the more new DNA is synthesizing, which ultimately mean you have a higher expression of your target mRNA. Of course, the expression of your target mRNA has to be compared with the expression of housekeeping mRNA expression.

Check this link for general info:

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[Sr_08]

Thanks. The link was really helpful.
How does one design primers for the q RT-PCR?
I mean how do I start? Sorry for sounding dumb but, I am a novice in this field....

[luisillo]

As far as I know, it's the same as conventional PCR primers, I mean, the same primers can be used for both conventional PCR and for qPCR. As for the designing, you ought to have the sequence of your gene to begin with. Once you have it you can insert it in the Primer design software (i.e. Primer3) and it should give you some suggestions of primer sequences.

[Sr_08]

Thanx. I have the sequence of the enzyme whose mRNA levels need to be quantified at different induction conditions. But, based on other published work; it just not the enzyme itself but, other genes that are involved in its expression.

So my guessing is until I have the whole cassette of all the genes involved in the expression process doing a experiment of such a kind is going to be of little help right?

[luisillo]

It depends. Measuring how the induction affects the enzyme's mRNA expression is important unless it has been already done (I don't know much about the topic). In this case I think it'll make you see if your induction conditions affect or not the expression of the enzyme's mRNA, and depending on your results, you might decide to study or not the expression of those other genes involved in order to know the mechanisms involved.

Considering this, I think the experiment with only the enzyme's mRNA IS important as a starting point, then again, qPCR is kinda expensive. My advice is: if money isn't a problem, then do it. But anyway, that's just my opinion, and as I said before I'm not very knowledgeble on this topic (induction). Guess you better discuss it with your boss first.

The best of luck.

[Sr_08]

Thanks! So I can use the primers which I designed to pull out the enzyme from the gene(gDNA) and do a qPCR (With RNA as a template) ??

[luisillo]

That might be possible if your working with prokaryotic organisms. In eukaryotic might be a little difficult because the sequence change: genomic DNA has introns which are absent in a mRNA sequence. When you design your primers you have to put the sequence you want to amplify and as I stated above, it might differ from gDNA to mRNA, which will ultimately alter the sequence of your primers and thus their specificity.

I guess you'll have to figure that question yourself based on your knowledge about the topic.

Again, I wish you luck.

[Sr_08]

Thank u for replying to my post! I have really learnt a lot in these few days!!
One more question,
So, cells in uninduced state (will they be the housekeeping mRNA genes?) can be used to compare
expression of the target mRNA ???

[luisillo]

Uninduced cells will be the control for the assay. I think it is important that you include that experiment. But, for housekeeping gene I mean a mRNA that is constitutively expressed in the cell, some examples are B-actin mRNA or ribosomal subunits mRNA. By comparing the constitutively expressed mRNA expression against your target mRNA expression you can tell if there are differences in you gene's expression within the same cell culture (or condition or whatever you prefer to call it). In this way you discard random variability between experiments.