I have rna for MCF-10 and MCF-7 ....the concentration of rna of mcf-7 is 884 ng/microliter with 260/280 1.76 .. the concentration of rna of mcf-10 is 668 ng/microliter with 260/280 1.91
I convert rna to cDNA after uniform the amount of rna to 2 migrogram
I check my cDNA with pcr with primer GAPDH
I got small different size of band between MCF-7 and mcf10 although they express the same gene ?
Hi Asma, it is possible that there are splice variants between the two cell lines, and that could explain the difference. I found something on it here.(http://uswest.ensembl.org/Homo_sapiens/Gene/Summary?g=ENSG00000111640;r=12:6533927-6538374;redirect=no)
If you really want to figure out if that is the case, you can design primers that flank each exon (I did this in my first publication "Paralemmin" if you want more information).
Also, it looks like your gel is a little off, and I'm not sure what lane is what. How did you clean up your samples after PCR? It looks like there is more primer left over in the first two sample lanes, and that could affect migration (as you can see that sample 4 looks slightly smaller than sample 3)...I'm assuming that the samples are in pairs? Also, did you normalize the amount of sample loaded into each lane? Depending on what buffer the samples are in, that could affect migration...if you are loading more volume of sample into different wells. If you normalize the concentration of the 4 samples in the same buffer and use the same volume of sample for gel loading, then I think the most likely cause could be splice variants.
Best,
Casey
Hi Asma, it is possible that there are splice variants between the two cell lines, and that could explain the difference. I found something on it here.(http://uswest.ensembl.org/Homo_sapiens/Gene/Summary?g=ENSG00000111640;r=12:6533927-6538374;redirect=no)
If you really want to figure out if that is the case, you can design primers that flank each exon (I did this in my first publication "Paralemmin" if you want more information).
Also, it looks like your gel is a little off, and I'm not sure what lane is what. How did you clean up your samples after PCR? It looks like there is more primer left over in the first two sample lanes, and that could affect migration (as you can see that sample 4 looks slightly smaller than sample 3)...I'm assuming that the samples are in pairs? Also, did you normalize the amount of sample loaded into each lane? Depending on what buffer the samples are in, that could affect migration...if you are loading more volume of sample into different wells. If you normalize the concentration of the 4 samples in the same buffer and use the same volume of sample for gel loading, then I think the most likely cause could be splice variants.
Best,
Casey
Hi Asma,
I would likely agree with what Casey has already stated about the migration of samples along an agarose gel. There are many things that could cause different migration even among samples of the same size including the amount of sample loaded into the well. Unfortunately, agarose gel electrophoresis is not a very accurate platform compared to other more sensitive methods. It could also be related to the percent gel you were using.
Sequencing is the better way to know for certain. The PCR looks relatively clean except for presence of the primers. However, purifying the products to remove primers and excess salts could result in a fairly clean sequence result. This way you could determine whether there are any splice variants.
the problem seems with the quality of ur RNA for one of the lines. A ratio below 1.9 is a sign of DNA contamination. You should extract fresh RNA from cells and redo ur experiments.
Hi Asma, I agree with all the answers above, but have another suggestion. Do you routinely DNase I treat your RNA? GAPDH has a large number of pseudogenes in mammalian genomes that are non functional and contain a number of exons skips, intron deletions etc. It may be that one of your samples has a relatively high level of genomic DNA contamination and you are preferentially amplifying one or more GAPDH pseudogenes rather than GAPDH cDNA. The easiest way to check this is to do a no RT control with your PCR amplifications. If genomic DNA isn't the problem, then a quick way to start to analyse the problem is a restriction digest of the PCR products, although obviously as Doug says sequencing is better. I wouldn't waste too much time trying to solve the problem. If its not genomic DNA contamination and restriction digest/sequencing does not give you an obvious answer, change your primers. Primers are very cheap and restriction digestion/sequencing are less so. I would design and order 3 more sets of primers and try the RNA from both cell lines with each of these. Hopefully, at least one set will give you PCR products of the same size from each cell line, you can then confirm the products with sequencing when they are the same size. Don't forget to BLAST search you primers to make sure they are specific to GAPDH and cannot amplify spurious cDNAs.
Dear Asma Future
I agree with Artur Burzynski. It seems that you didn't run PCR products properly and your gel might have been run shelvy. I don't know why you have protein contamination in your Ladder, If the first well represents the ladder. I strongly recommend you to treat your extracted RNA, befor converting it into cDNA. Subsequently rerun your PCR products in a high quality Agarose gel. Let the gel form well and then load PCR products according to the following proportions: 8 microlitr DNA plus 2 microlitr loading Dye or 5 microlitr DNA plus 1 microlitr loading Dye or 4 microlitr DNA plus 1 microlitr loading Dye.
Good luck to you
- Why does different sizes of GAPDH band appear though primer is same. cDNAs from different mice. Iam humbly requesting explanation from the experts of this specific research field.
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