Hi
I have a few questions about the extraction of RNA and RT-qPCR. I am aware that the real time PCR is normally run on more replicates of the same sample. I was wondering how it is better to prepare these replicates. In particular, if it is better to use the same RNA sample in more replicated PCR reactions, or to perform more RNA extractions of the same biological sample and, therefore, use them as replicates.
Also, I tried to extract RNA from bacteria but I am not sure that I managed to get it. I used the RNeasy mini kit. At the end of the procedure, I measured the concentration of RNA with nanodrop. It was about 180 ng/ul. Anyway I also measured the DNA concentration (to see possible DNA contamination) and I found that the DNA was present at a concentration very similar to the RNA..is it possible that the nanodrop is measuring the same thing?I changed the settings between the DNA and the RNA measures, so I am quite confident to have set correctly the instrument. How can I measure the two nucleic acids separately?
In another case, I treated 1 RNA sample with DNAse I after the RNA extraction. I thought that the sample was contaminated by DNA because of the nanodrop results. After the treatment I measured again the DNA and RNA concentrations and I don’t have so much idea of how to interpret the data. I attached a picture of the plots I got. I measured the DNA and then the RNA concentration in the same sample and again I suppose the nanodrop is measuring the same thing. Also, the curve does not seem the typical curve of the nucleic acids..does it? Is this due to the contaminants in the DNAse reaction (buffer used, enzyme etc…)?Can you help me to understand what went wrong?It is the first time I work with RNA.
Thank you very much
Dear Silvia
If I understood You correctly, there is a misunderstanding of nanodrop principle. The maximal absorbance of both DNA and RNA is 260nm, so nanodrop cannot segregate them. The parameters that are allowed to select affect the formula that convert OD to concentration. So after RNA extraction I would run a gel to see if Your RNA is visibly contaminated with DNA, and if yes (very likely in my experience), treat Your samples with DNAse with following cleaning and re-precipitation. Then You can be sure that everything that nandorop measures at 260nm is RNA. Regarding Your attached results, I would say that Your main problem is salts contamination (230nm). That shows Your 260/230 ration as well. Try to get it close to 2.
I see. Thank you very much Albert.It was very helpful. Is there a specific protocol I can follow to clean the RNA samples and re-precipitation?Do I need a kit for this?
Thanks
Dear Silvia
I am not sure that You can successfully clean Your current samples, but You can try. For this just add an equal volume of isopropanol and centrifuge the probe. If my assumption about salts is correct, You will get a pellet (can be invisible). Then add 70% ethanol, shake, centrifuge, repeat. And last, dissolve in TE buffer or water, end try to measure on nanodrop.
The problem is that during this cleaning You will lose some RNA, and if You do not have much - You will lose almost everything. So, I would perform extraction from the beginning. As You said this is the first time for You extracting RNA, and in my experience the 2nd and 3rd extractions are usually much better. However, if the results will be bad again, try to change a kit, or perform manual extraction (I personally use usually LiCl-based). Another general suggestion for kits - do not use too much tissue, it can overload a column and lead to lower yield compared to those that can be extracted from a smaller amount of tissue.
In case of RT-PCR , generally it is recommended that in one time, you should have triplicate of your one sample.The entire expt. you do three times at different time points.so, sample replicate and experimental replicate both are equal important.One is for expt. validity and reproducibility and another for sample handling and skill of your hand.
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