mRNA Expression - How to observe mRNA expression of gene. (Oct/16/2008 )
Hii i am Amit
i am working on expression of mRNA but i have some problem regarding mRNA expression.
can you help me
please send me protocol for mRNA expression. i have this problem since last six month.
i have done cDNA amplification and PCR of cDNA but the problem is there is no difference in band. even in known gene which are differentially express.
i hope for your help
with regards
Amit singh
amitsingh.aiims@gmail.com
Did you run couple with house-keeping gene such as actin-B or GAPDH?
Are your bands too saturate? The saturate bands cannot be measure the difference.
Are your bands too saturate? The saturate bands cannot be measure the difference.
Thanks for your reply
i am using 16sRNA as a control.
can you send me the protocol which is suitable for the mRNA expression.
with regards
Amit
You are doing a PCR or a Real time PCR???
i m doing PCR
When u say u notice no difference, what do you mean ? the band intensity?
And since u denied that you\'re doing Real-time how is it actually that you did the quantification?
merely a typical PCR and comparing the band as u noted earlier i doubt will help in quantification but you might have some fancy tools.If u started off with different amount of cDNA ( as per your \"differentially expressed\" statement ) , PCR will proceed probably at different rate in the beginning, but since u provided them with excess primer and excess dNTP, it reached its Max output once it used up any of the reagent. and the cycle when this happen is hard to tell.
And since u denied that you\'re doing Real-time how is it actually that you did the quantification?
merely a typical PCR and comparing the band as u noted earlier i doubt will help in quantification but you might have some fancy tools.If u started off with different amount of cDNA ( as per your \"differentially expressed\" statement ) , PCR will proceed probably at different rate in the beginning, but since u provided them with excess primer and excess dNTP, it reached its Max output once it used up any of the reagent. and the cycle when this happen is hard to tell.
Yes i am talking about band intensity. i am not doing real time PCR. and i am using same quantity of RNA for cDNA synthesis. and cDNA is diluted with 70ul nuclease free water. then i used 2ul cDNA for PCR amplification (25ul reaction) and 20ul loaded in to agarose gel for quantification. after all i didnt got any difference. some bands are very faint but all are with same intensity.
can u tell me general procedure which is followed for the mRNA expression of genes.
And since u denied that you\'re doing Real-time how is it actually that you did the quantification?
merely a typical PCR and comparing the band as u noted earlier i doubt will help in quantification but you might have some fancy tools.If u started off with different amount of cDNA ( as per your \"differentially expressed\" statement ) , PCR will proceed probably at different rate in the beginning, but since u provided them with excess primer and excess dNTP, it reached its Max output once it used up any of the reagent. and the cycle when this happen is hard to tell.
Yes i am talking about band intensity. i am not doing real time PCR. and i am using same quantity of RNA for cDNA synthesis. and cDNA is diluted with 70ul nuclease free water. then i used 2ul cDNA for PCR amplification (25ul reaction) and 20ul loaded in to agarose gel for quantification. after all i didnt got any difference. some bands are very faint but all are with same intensity.
can u tell me general procedure which is followed for the mRNA expression of genes.
Real-Time PCR or microarray. procedure? as per manual.
Hi Amit,
Sorry if I missed some details somewhere, but here are some questions I have for you:
How many cycles are you running? (Maybe you're running too many)
Do your gene of interest primers span exons? (Maybe you're picking up genomic DNA contamination instead of RNA transcript)
I don't understand about some bands being "very faint" but still being the "same intensity" as others? Are you detecting with ethidium bromide/UV light? Or with SybrGreen/STORM?
How do your controls look? (16s bands, negative controls, etc)
It sounds like you're doing the basics for detecting mRNA expression:
Extract/purify RNA
RT reaction to produce cDNA
PCR reaction to amplify gene of interest and control transcripts
Sounds like it just needs some optimization.
OK, so in your experiment you are looking at PCR product from a mRNA by examining the intensity of bands produced at the end of a PCR.
PCR works by amplifying the product in an exponential fashion per cycle i.e. 1,2,4,8,16,32...etc., until the reaction is limited by some factor which in this case is usually the amount of dNTPs or the number of primers available. If you run a standard PCR with 25-30 cycles, you will have a massive number of product sequence at the end (approx 1*10^25 products), even if you start off with a single copy of the RNA in the starting reaction. Because of this you will not see much difference in the end intensity on the gel, and, from the signal produced cannot deduce anything.
This doesn't even take into account that Ethidium bromide (presuming you are using it) actually has a limited detection ability in a gel, no matter how much you put in. I think it is limited to about 200 ng/band, so not very sensitive.
If you want to look at it in a gel, what you need to do is at each cycle (or every 2-3 cycles), you need to take a bit of each reaction out and run them on the gel, plot the intensity on a graph and work out the reaction slope from there... this is the most basic form of real time PCR, which is what you really need to be doing.