I am doing qualitative RT PCR to detect the gene's expression in my mutant in comparison with wild type (expectedly the mutant must not have any expression because it has TDNA insertion) but every time I do PCR with Tubulin or gene-specific primers, the results fluctuate. The concentration of RNA is okay. Tm of the primers is 58 degree. What parameters should I adjust to get consistent results?
..what do you mean with "fluctuating"? Can you also describe your experiment in more detail? Amplicon size, RT-priming strategy, RNA/cDNA amount etc?
If you're saying that your samples vary in expression by 2-4 fold even for reference genes, this is entirely normal: such variation is common and is EXACTLY WHY you must always include reference genes. If every sample always had exactly the same amount of cDNA, and all your efficiencies of RNA isolation and cDNA synthesis were 100%, you wouldn't really need a reference gene at all. Sadly, efficiencies are rarely 100%, and are rarely consistent between samples, so you do need reference genes. Ideally two or three well-validated reference genes appropriate for your samples.
You must mean quantitative?
Normalize to several reference genes selected based on your sample. Maybe tubulin is not the best choice. But Norman Hafner is right, the info you have given is not enough.
Norman Hafner Leonid Ouchanov John Hildyard
I first extracted RNA of my mutant and WT and converted it to cDNA. I used 1 microgram of RNA from each sample for cDNA synthesis. The PCR was performed using Tubulin and gene specific primers (my gene's size is 1 kb). The concentration of cDNA in a 20 microliter PCR volume was also 1 microgram. When I did PCR for the first time, the Tubulin gave the exact sized bands and if we talk about my gene, there was no expression in the mutant and WT had expression. When I repeated the PCR for second time, some of the mutants showed band. I considered it contamination and optimized the conditions (by optimization means I used tips, PCR tubes, cleaned my pipettes, used a new tube of PCR mix, new primer dilutions) but this time there was again fluctuation (I mean the the mutants that showed band in second PCR did not show the band but the other mutants showed bands). The same problem was with Tubulin. The PCR conditions are as follows:
95°C for 3 minutes
94°C for 30 seconds
58°C for 30 seconds
72°C for 30 seconds
72°C for 10 minutes
I am hesitating whether should I trust the results of very first PCR or should I make cDNA again and repeat the PCR. I need your recommendations. Thanks a lot for your answers!
Leonid Ouchanov This is not quantitative RT PCR but qualitative RT PCR. I am concerned with the to check whether my mutant is knocked out or not. I mean if it is knocked out, there must be no amplification with gene-specific primers (and hence no gene expression) in mutant plants but there must be amplification in wild type.
Dear Tahmina,
I would suggest that you are at the detection limit for your PCR resulting in the observed fluctuations. Nevertheless, this is surprising because you put a large amount of cDNA into the reaction- 1 micro gram (?). At least your control gene tubulin should be stably detectable. For your mutant samples it may still be a contamination or you have a very low expression of the wt gene. Another problem can be the size of your amplicons. If they are ca. 1kb (?) and the RNA quality is low, this may cause a low amount of cDNA molecules with a size >1kb. So you may check the RNA quality, test another housekeeping gene and use another primer pair for your gene of interest to exclude a contamination with the actual PCR product.
Good luck!
If you're using 1ug of cDNA, and you're synthesising 1ug of RNA into cDNA, are you not using effectively neat cDNA?
If so....don't do that. Ever. Not only does neat cDNA contain a whole swathe of cDNA synthesis components (salts, reducing agents, random primers) that can inhibit PCR, it also wastes masses of cDNA. You should need nanograms, or tens of nanograms at the worst.
I suspect beta tubulin still works because there is MASSES of this message in your pot, so even with your PCR reaction inhibited badly, you still see a band. For your GOI, maybe not so much.
I do all my analysis via qPCR rather than 'qualitative PCR', because it gives me data that is every bit as qualitative, but also has numbers to support it, but still: I use ~4-8ng of cDNA per well and this allows me to robustly detect even genes of relatively low abundance.
Try diluting your cDNA 1/20 and then using a small amount of this in PCR instead.
I suggest to do optimization in tempreture and quantity you taked from primers and cDNA after checking concentration
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