A and B genes were put in the MCS of an expression vector so they are transcripted in the same Ptrc promoter. After electroporation into Corynebacterium glutamicum I found that products increase. I am trying to detect the level of A and B gene expression. Any advice ?
RT-PCR using specific primers works the best. If your genes have alternate transcripts (splice variants), make sure you have primers that bind to exons present in all the transcripts or for specific transcripts that you want to measure expression of. If you want to do high-throughput assay to test several genes, you can do a high-tech slightly costlier technique called RNA Seq also referred to as Whole Genome Sequencing or you can go for microarray analysis. More info for these techniques can be found by a Google search. There are companies or core facilities that do RNA seq or microarrays for you. Good luck!
First question is to be asked that A and B genes are foreign genes or native genes. If these are native genes, the Real Time PCR can give change in expression level compared to control Delta CT value. If it is a foreign gene, Western blot can give idea about differential expression between two genes and use of empty vector would serve as control.n
RT-qPCR, but be careful to select the proper reference genes for normalization.
Or you can use Western Blot and check the intensity of the bands, also in this case you need to have proper reference genes for the normalization.
The easiest way to study gene expression is the quantitative PCR if you have a real-time PCR instrument available. Otherwise, you can use the traditional Northern blotting.
Northern blotting would be my suggestion. You check the levels of transcripts. Real time PCR is also an option.
RT-PCR using specific primers works the best. If your genes have alternate transcripts (splice variants), make sure you have primers that bind to exons present in all the transcripts or for specific transcripts that you want to measure expression of. If you want to do high-throughput assay to test several genes, you can do a high-tech slightly costlier technique called RNA Seq also referred to as Whole Genome Sequencing or you can go for microarray analysis. More info for these techniques can be found by a Google search. There are companies or core facilities that do RNA seq or microarrays for you. Good luck!
Just go for RT-PCR.. If it is possible use TaqMan probe instead of Syber green. Compare with mock (unrelevant or empty vector) transformed bacteria...
Depending on your experiment: you may first have to decide whether you want to see gene expression in terms of protein product or in terms if mRNA product or both (Gene-->transcription--> translation).
qPCR/RTPCR when done using the right normalization genes is perfect for mRNA expression levels
Western blot is semiquantitative but if the protein is soluble prt: ELISA can also serve you well.
If you need to find the best reference genes for your RT-PCR i would suggest you to have a look at :
http://www.refgenes.org/.
If you need some help to use this software, please let me know. Cheers
Why are you interested in gene expression, do you want to know if your organism producers RNA, or protein from that gene? In most cases you would want to know if the protein is there, surely?
qRT-PCR - this will give you an quantity of mRNA levels, if you want to look at protein expression I might suggest a western or ELISA
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