Hi, I extract RNA from leaf under normal & different stress condition, and synthesize cDNA but when I check its expression level with tubulin , untreated leaf give me normal result as show in figure the 1st band, but stress give me bad result after PCR. Is it RNA? OR some problem in my c DNA. Please its urgent. Thanks
Hi Bushra,
Is this RT-PCR? Yes, it is strange, tubulin is an internal control. With a normal RT-PCR result, it should show one band.
You need a noRT control in your RT-PCR experiment (a sample you treat the same without putting the RT). If you see bands in a noRT control, it does mean that your RNA samples are contaminated with DNA. Even in the case you did a DNase treatment you have to do a noRT control.
This looks like banding associated with programmed cell death that can result from severe stress, but I'm not sure why that would affect tubulin cDNA. You might need to dial back the stress treatment, or take samples at earlier time-points to preserve RNA integrity and observe the physiological response.
Personally, I don't think it is 'Apoptotic DNA fragmentations' due to programmed cell death. The reasons are 2 folds:
(1) The fragmentation 'fragments' should be observed from the whole lane on the gel [see attached picture #1 for an example. Lanes: Apoptotic DNA fragmentation, visualised by the DNA laddering assay (left lane). A 1 kb marker (middle lane) and control DNA (right lane) are included for comparison.]. Also, picture #2, showing 'fragmentation' on a gel, indicating GA induced apoptotic death of cells. However, in the gel attached by Bushra shows that the 'bands' only concentrate at the lower part of the gel.
(2) Apoptotic DNA fragmentation is due to the activation of endogenous endonucleases which subsequently cleavage chromatin DNA into inter-nucleosomal fragments of roughly 180 base pairs (bp) and multiples thereof (360 bp, 540 bp etc.). However, during total RNA extraction for RT-PCR, the majority of gDNA has already been removed by DNase digestion (From other questions she posted, I believe Bushra has done DNase digestion.).
In my case what is real reason of this? Why multiple bands show here in stress conditions, why not show under normal condition grow leaf?
1. That is a good question! I suspect it may be just some 'unspecific' PCR bands, but I am not sure. Do you have another pair of primers to amplify this gene? You can perform a few more PCR with other primers to see what the results are. I would also try using a pair of primers to amplify half of the gene and another pair of primers to amplify the other half of the gene. To see whether it is only a band amplified or multiple bands amplified.
2. From your gel, I saw you had run 3 samples: (1) Untreated (control), (2) and (3) with treatment. Were the (2) and (3) TWO independent total RNA isolation samples, or they were the same RNA sample, and you just did RT-PCR twice ('duplicates')?
I already check with new tubulin primers, but same result. These are two different samples of different treatments with different time points. May be my cDNA degraded or contaminated thats why.
Did you isolate these 3 RNA samples at the same time. I meant the chemicals you used and procedures/protocols were not different when you isolated these 3 RNA samples.
By the way, when you isolated RNA from these plants, were those 'treated' plant (tissues) look green and healthy? Comparable to the control plants??
Yeah procedure is same and when i collect it, they are not so healthy
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