I have isolated RNA from a Cyanobacterial culture using TRIZOL. The concentration of RNA is around 500ng/microL, and purification parameter as well as gel run indicating good enough the quality of RNA. I used hexamer as primer to synthesize cDNA from that RNA using Invitrogen kit. Then, I made PCR using designed primer from the cDNA to test whether the cDNA of my target has been synthesized or not. Unexpectedly, I have not found any band except control which is also not correct in size. Anybody has any idea about the solution to above problems?
Rezaul, i think you need to check few thinks here.
First of all you need to check whether the RNA is getting degraded or not and its storage. Make sure the water you are using are all RNase free and the primers you have designed are all designed based on the cDNA template.Because i have used cDNA first strand synthesis kit of invitrogen to obtain my result from mammalian cell line, and it has produced fine result for me. I think there must be some small technicall glitch which is preventing you from getting the result. So check the above two things, hope your problem will be sorted out.
Best of Luck !!!
Dear Dipon, Thanks for your suggestion. I have used DEPC treated water in all the perpose of RNA isolation. To be sure about degradation, I have made gel run which is showing clear three bands. Before using this RNA for cDNA synthesis, I stored it at -70 for two days. I haven't check again whether the RNA has degraded upon storage or not before starting cDNA synthesis. However, I would test it in my next experiment. Considering your second suggestion of primer designing, I am using Cyanobacterial RNA where there is no intron as you found in mammmalian cell. I have designed primer for amplicon of between 100-200bp in size. In my lab, many students are using this same kit to make cDNA from mammalian cell and like you, they are highly sucessful. Do you have any other idea to solve the problem.
Thanks again for your kind response.
Khan
It may have to do with the cDNA prep itself. If the temps were wrong or you added the reagents in an improper order this will effect cDNA synthesis.
Maybe your RNA has a lot of secondary structures. Some RNAs require preheating in 100 degrees celcius along with the random primers, and then addition of the rest of the cDNA reaction ingrediants with the protocol for it, to overcome this problem
Did you run RNA on a gel? 1st thing that I would do is that & check wheter the RNA is degraded or not. If not you are good to go. Then the problem arise in cDNA preparation. Better to go back to drawing board & check one by one. Good luck.
Thanks every body responsing on the problem. Especial thatnks to Shachar Raz giving an extraordinary insight about the problem.
Did you use random hexamers (you just say hexamers). I find these often work best. Also how long is you PCR product. Often I have trouble with things longer than 1.5 kb but I can get them to work if I do the PCR as two reactions, making the 5' and 3' halves separately. Suggesting that some of the RNA is probably nicked.
@Kevin Nash, Thanks for your suggestion. Yes, I have used random hexamers. The amplicons for which I have designed primer for PCR are between 100-150bp and preferably from 3' end of the target gene. My target is to use that cDNA for qRT-PCR. After making cDNA, I have just made a normal PCR using that primer to see if the cDNA is synthesized or not. Unfortunately, I haven't found any bands after electrophoresis even for control.
Your suggestion of designing primer for sequence from two seperate end is very nice. I would follow that.
Thanks
Khan
How much of your RNA is mRNA in the preparation? Was the RNA run on agarose to see the purity ? Why not use a oligo DT column to get poly A mRNA for better cDNA synthesis
I agree with all the above. You first must check the RNA. The most of the kits work robust. I've noticed, that even if you put water or blank in your tube, the kit gives you results with good absorbance. But in qPCR you got no prodyct.
Hello together.
@Siva: oligoDT column purification will not work with cyanobacterial RNA as there are no poly-A tails like in eukaryotic cells.
@Dipon Das: RNA is definitely less stable than DNA and preparing cDNA immediatly is certainly not a bad idea. It is no problem, however, to store RNA at -70 to -80°C for several months.
@Khan: 2 days of storage after your quality check (gel etc.) will not have changed the RNA, you do not need to run another gel.
Have you thought about the possibility that the RNA you wanted to see, is actually not expressed under the conditions you tested? have you tried amplifying a housekeeping gene from your cDNA prep? You should do that as a positive control, to see whether or not you have cDNA at all. Take rnpB, for example, that always works.
All the best, Ulrike
@Ulrike Pfreundt , many many thanks for your kind reply. I have taken rnpB as positive control. However, when I made PCR, I found this gene is showing a band but not correct in size. Later on, I made PCR again with genomic DNA to be confrim that the primer is OK. Unfortunately, I haven't found any band except rnpB but, like cDNA, the uncorrect size. From these information, do you think the primer I have designed has some problem or the problem is in cDNA generation?. Please give me some suggestion if you can.
Thanking you
Khan
If your primer pair did not yield any product when you did the PCR with genomic DNA, then I suggest your primers are indeed the problem.
Would you like to send me your primer sequences and tell me which genome?
It is clear that when you haven't got any amplification with genomic DNA itself the primers are the culprits which need to be relooked into.
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