If I do cDNA synthesis with SuperscriptIII using 2 mg of total RNA in 20 ul reaction, do I need to quantify cDNA by nanodrop and optimize concentration for RT-PCR, or can I just use 0.5 ul of cDNA mix straight away?
2 mg of RNA per 20 uL RT reaction is way too much RNA, it will shut down the SSIII enzyme and you will get no (or very little) cDNA. 1 ug of RNA per 20 uL RT reaction is what you would want to use. Your 2 mg = 2000 ug (so you are planning on using 2000X too much RNA in your 20 uL RT rxns). A good RT rxn contains 50 ng RNA/uL.
Also, you can't quantify cDNA by NanoDrop, as you would be quantifying all dNTPs and RNA [RNase H-digested and undigested] and cDNA in the final reactions - giving no indication of how much [just] cDNA you have. This mistake is also confounded further by the frequent (also erroneous) practice of zeroing the NanoDrop or Spec with water instead of RT reaction components minus the RNA.
Typically, before RT, you would quantify your RNAs by spec. or NanoDrop, then add the exact same amount of RNA per RT rxn (e.g.1 ug RNA per each 20 uL RT rxn), then dilute the resulting cDNA samples at least 1:10 prior to RT-qPCR. But, you would want to do a dilution series of your typical cDNA to see where the viable log-linear range is beforehand so that you don't just use some blind dilution (e.g., 1:10) without reason attached.
Checking the RIN number (RNA integrity number) of your RNA beforehand on a Bioanalyzer 2100 is very important to show that you have high quality RNA prior to RT.
Typically, you would want RIN values of 7.5 or greater.
Another point: your acronym "RT-PCR" -- I am not certain whether you intend "real time-qPCR" [by machine] or "reverse transcription-PCR" by gel analysis...
2 mg of RNA per 20 uL RT reaction is way too much RNA, it will shut down the SSIII enzyme and you will get no (or very little) cDNA. 1 ug of RNA per 20 uL RT reaction is what you would want to use. Your 2 mg = 2000 ug (so you are planning on using 2000X too much RNA in your 20 uL RT rxns). A good RT rxn contains 50 ng RNA/uL.
Also, you can't quantify cDNA by NanoDrop, as you would be quantifying all dNTPs and RNA [RNase H-digested and undigested] and cDNA in the final reactions - giving no indication of how much [just] cDNA you have. This mistake is also confounded further by the frequent (also erroneous) practice of zeroing the NanoDrop or Spec with water instead of RT reaction components minus the RNA.
Typically, before RT, you would quantify your RNAs by spec. or NanoDrop, then add the exact same amount of RNA per RT rxn (e.g.1 ug RNA per each 20 uL RT rxn), then dilute the resulting cDNA samples at least 1:10 prior to RT-qPCR. But, you would want to do a dilution series of your typical cDNA to see where the viable log-linear range is beforehand so that you don't just use some blind dilution (e.g., 1:10) without reason attached.
Checking the RIN number (RNA integrity number) of your RNA beforehand on a Bioanalyzer 2100 is very important to show that you have high quality RNA prior to RT.
Typically, you would want RIN values of 7.5 or greater.
Another point: your acronym "RT-PCR" -- I am not certain whether you intend "real time-qPCR" [by machine] or "reverse transcription-PCR" by gel analysis...
In addition to the above, ample dilution of the RT rxn [cDNA] prior to qPCR or PCR is needed to help undo any inhibitory capacity of the RT rxn components themselves. E.g., using 10 U/uL of SSIII during RT can hurt the PCR since the RT enzyme itself (even denatured) can still bind nascent amplicons during PCR; giving sporadic results.
Thus the need for ample dilution of cDNA prior to PCR and/or only using 5 to 8 U/uL of SSIII during RT. Often times in these things, less is more - within reason coupled with the empirical wisdom gained over time by performing these lonely rituals numerous times with your own hands and your particular samples, amidst your own unique set of circumstances...
Last point: the way you isolate your RNA (and the sample type from which you isolate the RNA) can also introduce inhibitors into your RT reactions and thereby kill them off early on as well. So, clean, high-quality RNA is the best foot to start off on: always.
Thank you Jack,
* My typo, I meant 2 ug of total RNA for RT in 20 uL reaction (100ng/uL)
In this case if I use undiluted 0.5 ul of cDNA for qPCR, supposedly I take 50 ng, which seems reasonable, right? Same 0,5 ul of undiluted cDNA contains only 5 U of SSIII which is still non-inhibitory. (1 ul of c=200U/uL used in 20 uL reaction).
*About dilution.
I will do primer efficiency with log dilution from 100 ng - 10 pg. Giving proper efficiency I may proceed with undiluted 0.5 uL of cDNA for my qPCR rxt, or I am not getting the point for essential cDNA dilution?
*Do I have to use primers diluted in Tris-HCl (pH=8), or is it advisable only if unspecific peak in melt curve is present?
*I see the concern of measuring cDNA by nanodrop, but given the same initial conditions for RT, it will at least relatively verify the same cDNA content for sample and control. e.g If I use 2 ug of each sample and control for the same 20 uL SSIII reactions. eventually I should get the same amount of cDNA but nanodrop for both of them. (Even if it is not pure cDNA measured against water).
*Regarding RNA quality it is a must, I agree. But do we somehow check cDNA quality?
As for Ag, I'm surprised to see this news just now. It has been known for quite a while and even some mechanisms were studied back to 20th century. I even did similar studies of AG with antibiotics few years ago http://pasteurorg.ru/images/infektsiya-immunitet/vyp2/abstract_Krat_Afonina.pdf
Perhaps it is the ugly side of the medical-industrial complex that wishes not for the best of medicines to be available. Apparently Ag was used to disinfect meat carcasses until about 1938 when one too many blue-gray people resulted after years of exposure - but no deaths associated. Anyway... on the qPCR stuff above:
In my experience (back to 2001), RT reactions containing more than 50 ng total RNA/uL fare poorer and poorer in cDNA yield. And measuring with NanoDrop will still tell you nothing --you will see that if you measure your RT reactions before and after RT you will get very nearly the same reading both times (the universe inside the tube hasn't changed, (the same 260nm-absorbing players are there but just in a different arrangement/incorporation). But, a Qubit, fluorescence-based system can get you what you want (~$5000 system) using Oligreen (or other dyes that only bind single stranded DNA like cDNA). So, with enough money, there is a way to get what you want -- but NanoDrop is definitely not the way to go for the answer you seek on that...
also check this out:
http://qpcrupdate.net/librus-norm-2006.pdf
Addressing your question:
"*About dilution.
I will do primer efficiency with log dilution from 100 ng - 10 pg. Giving proper efficiency I may proceed with undiluted 0.5 uL of cDNA for my qPCR rxt, or I am not getting the point for essential cDNA dilution?"
Yes - it is very important to do the dilution study (ideally a 15-pt serial 1:2 dilution study to see very precisely where inhibition lets up) to determine how concentrated you can use your cDNA in the qPCR application. You will often see that the most concentrated sample inhibits the qPCR (maybe even the 2 or 3 most concentrated samples). Once you find out the dilution range within which you get no inhibition and high efficiency, you would dilute your individual samples to the same point within the early part of the standard curve for final sample measurements. Using your samples at full strength would be contraindicated by the dilution study if it revealed that doing so inhibits the qPCR.
Regarding quality - it is most important to check RNA quality before making cDNA.
TE pH 8.0 for storing primers is a good idea since DNA is more stable at pHs above 7 and a little EDTA in there helps offset any divalent cation-dependent cleavage of oligos by inadvertently/environmentally-introduced DNAse.
Hi,
If you normalize the RNA concentration (our lab we use 1 micrograms or 0.5 micrograms of RNA concentration) for the synthesis of cDNA. you no need to measure the cDNA concentration. you can dilute the cDNA to 1:20, From that you can use 4 microlitre for 20 microlitre SYBER reactions. You will get better peak.
Thank you,
I feel using 1 ug to 2 ug is good enough, If i have not got so good quality of RNA i prefer to use 2 ug. more than this i never got good results, 2 mg is too much. Quality of RNA depends on tissue to tissue , so once you decide how much RNA you are going to use for cDNA, use the same quantity for that tissue type for all future experiments to get good comparative data.
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