For the RNA precipitated, most concentrations are around 10ng/ul with some even lower than 10ng/ul. The 260/230 ratios are also poor, most below 1. Can cDNA synthesis still work for these samples?
Hi Alisha, this entire Pandora cannot be sum up in a single line.
I have been on these kits since long, especially Applied Bio Systems High Capacity RNA to cDNA kits (50 and 1000 reactions both). If you have the 1000 reaction kit, then as per their protocol you have to take 10 uL of RNA regardless of its concentration. I found some technical difficulties with this kit, so I do not prefer to work with it, but their 50 reaction kit allows a maximum RNA amount 9 uL, so here you can calculate as per your desired target of RNA input. So for example, if my RNA is 50 ng / uL so I will take 4 uL of RNA to make it 200 ng / uL, which is quite safer for cDNA synthesis. These kits will usually give a 20 uL cDNA total volume in last, which you need to keep in mind. These kits allow 20 ng – 2 ug RNA and the final cDNA product is near 7 kb, also the enzyme used is really good. So it looks to me no problem in your case.
Just remember too that never apply excessive DNAse treatment to your RNA samples, as it is a known fact that any method of gDNA removal cannot occur without any loss (though even minute) in RNA quality and quantity and in your case it is surely not a good approach nor desirable.
The low RNA concentrations which you are having, I also got values like this several times, so in such cases you can even use 9 uL RNA (maximum allowable limit by the 50 reaction kit). You would be lucky if you have less GOIs and less samples to be tested in your qPCR with that low quality RNA, as you need then more cDNA, which means ultimately more RNA sample, otherwise what I do is to use more amount of RNA and calculate the amount of enzyme and buffer of cDNA kit and prepare a good volume of cDNA just one time and freeze it. By this way at least you can avoid the steps involving more degradation of your already low RNA in terms of repeated freezing then thawing and exposing it to working environment and preparing cDNA again & again etc. I prepared many times 60 uL cDNA total volume, so in this I used maximum of 27 uL RNA (which is 3 times, 9+9+9 uL).
In case of fragmented and degraded RNA must try to use SuperScript IV, as mentioned by Sir Laurence. Also, in cDNA (RT) step it is better to use RNAse inhibitors (1 uL per reaction) also available by Applied Bio Systems. Then if possible, just after the RT step use the cDNA purification kit (Ambion, AM1756) to purify your cDNA further and use it for your qPCRs.
Always remember that cDNA readings are never trustable by Nanodrop or any UV based spectrophotometer mean, so never quantify your cDNA, otherwise you may get a shock, so best way always is to quantify the RNA concentrations you want to adopt in the beginning of cDNA-RT step, and follow the same concentration values which you have used in first attempt later also to for the preparation of cDNA from these RNA samples. In different scenarios, I have used calculated RNA ranging from 200 ng / uL to 1000 ng / uL in these cDNA kits and it worked.
If your cDNA gives good readings on qPCR for your reference or housekeeping gene(s) so it means your cDNA is really ok and you are green to go. One common well prevailed rule of thumb is that only 20 % of the RNA will convert into cDNA( if assuming a 100% conversion), so these low RNA concentrations, which you have mentioned, in my opinion it is better to take 9 uL of RNA if you are making a 20 ul cDNA reaction.
In last, revise your RNA extraction protocol with extreme care and dedication, if you are using column based strategies so try to switch on magnetic beads protocols, if possible or improve your technique. Regards…and sorry for your time…
Laurence, I was extracting RNA from cardiomyoctes cultured on an RTCA plate, however the cell density per well was low, so I wasn't expecting a large concentration of RNA, I was just hoping maybe the little RNA extracted could be used for down stream applications. Thank you both for your answers.
Thank you Laurence, this has been really useful and depending on whether we decide to repeat the experiment or not, I will take the advice on board and try it. We are using RTCA plates which only have a density of 24,000 cells/well at most (500,000 cells/well was the recommended).
Dear Laurence Stuart Dawkins-Hall
,I did find your cDNA synthesis protocol using Superscript III with both oligo dT and random hexamers quite promising. But I would like to know where was this protocol adapted from? Like I do not see any paper citations. If I am adopting this method to prepare my cDNA how should I cite this protocol?
Thanks :)
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