Dear all, I want to determine gene expression of Glycerol-3-phosphate in Pieris brassicae. Nucleotides of the molecule is 621. I designed two primers one copies 600 bp another 243. Which do you suggest? One of my colleagues believe for gene expression, product must not produce more than 200 b. What is your suggestions?
To check if the gene is expressed by PCR (I suppose so since you are talking about primers) the length of the PCR product does not matter.
To measure how strongly a gene is expressed will require some quantitative PCR method. Here a suggestion will depend on what method you will employ. For real-time PCR, for instance, shorter products are preferrable (because this technique relies on equal and constant amplification efficiencies what is best assured when the reaction is set up for reaching almost optimum efficiency, what usually requires shorter products). Product lengths may go down to as little as approx. 100 bp (much shorter products will become more difficult to be distinguished from primer-dimers and will have little fluorescence yield [-> bad signal-to-noise ratio] when dsDNA-specific probes [e.g. SYBR Green] are used).
Dear Arash, I agree with Jochen... If you want to see whether your gene is expressed or not, conventional RT-PCR is Ok and the length of the amplification product does not matter very much (Do not forget you have to synthesize cDNA from an RNA preparation prior PCR amplification). However, if you are looking for something more quantitative, you should perform RT-qPCR and you should be very careful in choosing the primers.
Good luck...
Ricardo
Ricardo is correct. If you want to quantitate expression (which means you will be doing qPCR), make sure the primers you design give you an amplified product ~200 bp. Otherwise, if you just want to simply look for changes that are easy to see visually (less quantitative, but a good place to start before you go ahead with qPCR), your product size would matter less. Good luck!
Most reliable is u can go for sds page with suitable positive and negative controls
The qPCR is one of the most sensitive methods for studying gene expression. You need shorter products (100-150bp). However, you first need to optimize the primers and then check theri efficiency. Finally, you can use Livak Method (2^-ΔΔCt) and perform the analysis. It is an easy way to study just genes. But you should keep in mind that western will provide more details about the proteins. as the gene expression does not mean and protein expression.
Beside what our colleagues suggested, I am wondering if GAPDH can get gene expression? because this gene is supposed to be house keeping gene. I worked on steroids that not cause this gene to be expressed. if you know another steroid causing expression of this gene, them please let me know. All the best.
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