After trying different protocols I was able to extract RNA from old olive tree leaves with a phenol / chloroform : IAA protocol. Nanodrop measurments of the samples gave 260/280 rate ranging between 1.25-1.42 and 260/230 from 1.04-1.55 and a content of 100 - 200 ng/ul. I proceeded creating cDNA using 200ng of RNA template for a 20 ul cDNA volume. Then a qPCR was performed with the Kapa SYBR FAST qPCR Master MIx (2x) with the use of 1ul of the created cDNA for a 10ul reaction. The amplification plot looked like the attached. I am actually inexperienced but i was told that the refrence gene i am using (actin) is showing up late (30-35+ cycles)
I think that there is probably a phenol contamination in my samples and that is the cause for this bad qPCR. But i am also thinking about the quantity of the RNA that was used. Maybe it was too little, explaining the late amplification or too much taken the fact i didn't dilute my cDNA. Thank you very much in advance!
I saw that you did not have a good quality of RNA. Good RNA quality is usually signified by A260/A280 of around 1.8-2.0 and A260/A230 of around 2.0. Then, also I saw that you convert to cDNA using a low amount of RNA. Typically, you might want to use 1 µg to convert.
So, before performing qPCR, I suggest to check on the quality of RNA. Simplest method, which you already did, by spectrophotometry or alternatively by gel electrophoresis by observing the presence of other RNA components, i.e., rRNA, since you are probably isolating total RNA. Some google search will lead you to what is necessary to do and what will you expect from each method you use. Good luck!
Dear Vasiliki Kavoura per se the amount you used per qPCR shouldn't be a problem imo, in facts it is quite a lot (you may have to dilute it more). That is assuming the concentration measurement is ok and RNA intact; did u heck that there is no DNA and the RNA is not degraded?
Otherwise, the most likely is purity as you say: Your 260/280 260/230 are quite low, so as you mention there could be contamination of possibly phenol and that would be a problem (not just for amplification but also Reverse Transcription. So your problem can be both cDNA synthesis and qPCR.) If you can try to cleanup the RNA more or use a different protocol maybe it would be better (check this recent study for difficult plant samples:
Article DNA-free high-quality RNA extraction from 39 difficult-to-ex...
Maybe there is something helpful in here for your case.Apart from that, for the qPCR step I think it is good to dilute the cDNA more in general, and you can make a dilution curve to see if you have something that inhibits or just generally low levels. You are using a lot, for qPCR you can use a dilution of 1/100 or more depending on expression level and how much you have.
So you could make serial dilutions and use e.g. 1/20 1/40 1/80 etc etc and from the curve you can see where the amplification efficiency is ok (in this case e.g. 1/2 dilutions should correspond to 1 Ct difference for 100% efficiency. This might be a bit difficult with your Ct values being very high, though, which will give a lot of variation. Either way, best recommendation is to try and have better quality RNA.
Last, maybe you can include a positive control (some easier sample RNA, something you are sure will work and give good RT and amplification for actin, to exclude that there is any other problem in the process.)
Good luck.
Thank you all for your advice Win Sen Heng Robert Adolf Brinzer Daniela Liebsch ! I will follow your suggestions
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Extraction