Hello.
I'm doing cDNA synthesis with total RNA extracted from Escherichia coli O157:H7.
I got nano-drop result like the table below. I think the RNA concentration is too low.
But the volume of RNA solution is 50 ul and I think I can adjust the density of my solution to appropriate density to do cDNA synthesis using centrifugation.
My point is, after centrifugation(e.g., 10,000 x g, 20 min), discard supernatant and resuspend the pellet with 5 ul of RNase free water, I can make 10-fold more dense RNA solution and it will be appropriate to the next step for cDNA synthesis.
Do you think it can be made it? It's my first time of PCR, so I'm afraid if it won't work well.
Thanks for reading.
Hi JooWon Kim
I don't think you need to concentrate your samples .if you are planning to use 1ug of the RNA for cDNA synthesis reaction ,I think the concentration you have is good since the total volume is 50 ul (more than enough)especially the first two sample
if you are worried because of the low conc. of the last sample of RNA you can use 0.5 ug of RNA for the cDNA reaction ,and then for PCR you can use 1:5 dilution of the cDNA for PCR reaction .
and don't worry I think everything will go well.
Best
Hi
I would rather use the RNA as it is. Most kits allow to use at least 10ul RNA input which equals to 600-700ng. This should be sufficient. Repurification always has the risk to loose nucleic acid so first try without. In case you only look to analyse one or two genes I would consider the usage of a one-step PCR approach
Sven
Dear Sven,
I really appreciate to your help.
If I follow your protocol, is there anything I need to change from my own protocol?
I'm using real-time PCR to check the level of gene expression.
I think because the less RNA is used for cDNA synthesis, the more PCR cycle is needed. Is that right?
Hi
What is the Cq value your are obtaining and which RT kit are you using? In case you can share some more details we can try to find a good suggestion
Sven
Dear Low concentration is not an issue in science, you can deal with it also. One thing more, share some more information about your protocol and which type of kit you are using in your lab.
Kindly also updates us with the results. Are you getting your RT-PCR Results?
Shuja
I think there is some issue with our kits or something is going wrong within samples either they were degraded or something else.
Protocol that we had followed is genuine and correct.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA