I have repeatedly tested my primer efficiency values for several pairs and consistently get high numbers-- from 120-300%, with R^2 values at .99
I test six dilutions and have done this using dilution factors of 10 or 4 and starting amounts of 50 ng to 10 ng. I keep getting this same problem:
The first two or three ct values cluster very close together. The distance between ct values increases towards the last dilutions until it gets to what would be expected.
So it looks like I have inhibitors in my samples. They keep the most concentrated samples from multiplying like they should.
For this most recent data set, I performed a LiCl precipitation on the RNA, spinning it down and resuspending it in rnase free water. I then used 3 µg in a clontech RNA to cDNA ecodry premix kit.
Because the RNA was precipitated out (I could even see the pellet), I expect the only inhibitors present would be from the cDNA kit. If I were to treat this, though, I would lose confidence in starting RNA amounts for the expression study.
So, does anyone know about--
Hello Michael
I have never used that particular RT kit but will make 2 comments:
1. Does the kit recommend using 3ug of RNA ?: Most modern cDNA kits work well with 1ug of total RNA. By adding more RNA potentially you add more inhibitors present in RNA
2. In that regard, please note LiCl is the most specific salt you can use to selectively precipitate RNA (and not DNA and protein, unlike sodium and ammonium acetate) but you run the risk if you do not selectively desalt your sample of LiCl bleeding through into the final eluted (purified) RNA. LiCl potently a lot of enzymes including RT (see by way of example paper attached)
3. To more effectively desalt your sample and reduce LiCl contamination, having precipitated your pellet wash 2 x with room temp 70% ethanol. Further more, if you add 1ul of 1mg/ml to 20mg/ml glycogen as a carrier your pellet will be highly conspicuous when you precipitate and consequently you confidently manipulate; That is vortex to desalt. Basically, you add 1ul of glycogen (which is inert and does not therefore interfere with downstream processes) plus your LiCl and either ethanol or isopropanol (ethanol better) precipitate; remove supernatant; add 500ul of room temp 70% ethanol. Vortex to dislodge pellet; spin for 5 min to pellet RNA again. Repeat this wash. Spin again for 5 min. Remove supernatant. Spin for 1 min and remove residual spt from sides of tube with p10 tip. Air dry for 10min. Resuspend. Nanodrop then use your RT kit as before (but maybe using less total RNA if 3ug not recommended)
although it's been a while since I've done much PCR, I think you have to make sure there are not chelating agents in any buffers, also check for DMSO or sulfolane as although these help with strand formation etc in higher quantities they can inhibit the reaction. Promega has a good wee article on PCR inhibition with lots of references to look up too. It could be just a case of getting a kit that will tie in with the conditions in your lab, we found that some kits worked better for us than others, but talking to other folk they had different experiences. Good luck
Laurence, thanks for your answer.
In my overall LiCl treatment, I did wash with ethanol, though I hope it was enough.
50 µL of RNA sample, after being treated with DNase I, were added to 100 µL of 5 M LiCl. This was incubated at -80ºC for 20 minutes. Then there was a 20 minute 16,000g spin, followed by decanting, 150 µL 70% ethanol wash, repeat the centrifuge step, decant and dry for a few minutes, then resuspend.
I did not completely resuspend the pellet when washing nor did I use glycogen.
The kit allows for 1-5 µg. I will add the minimum as you suggest and see if there is a difference.
Thanks, your answer opened up some areas for me to inspect in my protocol.
Hello Michael. Try incubating for 1 hour at -20C instead of 20 min @ -80c. You might find a slight reduction in yield but at -80c you are more likely to bring down more of everything: RNA but also salts. If incidentally you add glycogen you will not lose any RNA even at -80C. Regardless repeat your 70% ethanol wash and preferably vortex of salt is a potential problem but I understand your reluctance to do that if you cannot see the pellet as effectively you are working blind ( although should still be fine; I have done this many times). As mentioned if you add glycogen you will see the pellet so will be confident to pulse vortex in order to dislodge and wash ( desalt) more effectively. Your recovery will also be 90-100% even at -20c ( instead of -80c). Good luck !!
Dear Michael.
I will agree for Laurence Stuart Hall ·comments to wards your problem solving
All cDNA kits I have tested result in PCR inhibitors in the cDNA. You can get rid of them by diluting your cDNA prior to adding it to the PCR, 10x should be the minimal dilution factor. You can identify inhibtors by looking at the shape of the amplification curve.
In higher dilutions of a dilution series, stochastic effects will effect the Ct-values (called 'Monte Carlo effect'), this has nothing to do with inhibition.
For cDNA synthesis, I never add the maximum amount of RNA into the reaction. I have answered this in one of your other treads:
"Did you ever do an optimization? If you titrate RNA into a RT-reaction, there might be a plateau where adding more RNA only has a negative effect downstream of your procedure (I did this experiments myself with different RT kits)."
What do you suggest for high primer efficiencies? - ResearchGate. Available from: https://www.researchgate.net/post/What_do_you_suggest_for_high_primer_efficiencies [accessed Jul 22, 2015].
To qualify Marcos comments I routinely use a 1:10 to 1:100 dilution of my cDNA. In addition, I usually perform cDNA synthesis in a 20ul reaction and like many other people I dilute this up to 50ul or 100ul before freezing away. It is this diluted mix that is further diluted >/= 10x in the actual expression analysis assay; The exact magnitude of dilution is based upon a preceeding standard curve
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