How to quantitate, to dose first strand cDNA ? - (Jan/11/2006 )
[quote name='smurray' date='Jan 24 2006, 11:08 PM' post='38422']
Hi,
I have a question about that last comment - so you are saying, its important to clean up the cDNA using spin columns or another method.
But its very difficult to quantify cDNA.
Did I get that right?
Which spin column method do you use?
thanks
Do you mean that I should to precipitate my cDNA reaction before to proceed to Real Time PCR ?
[/quote]
[/quote]
It is important to clean up the cDNA if you want to quantify it. However if you are doing simple RT-PCR then cleaning up is unnecessary
It is also important to clean up your cDNA following 2nd strand synthesis when proceeding to in vitro transcription
I use either the ChargeSwitch PCR clean up kit or RNA kit to clean up my cDNA, depending on whether it needs to be RNAse free or not
[quote name='John Buckels' date='Jan 25 2006, 02:33 AM' post='38510']
[quote name='smurray' date='Jan 24 2006, 11:08 PM' post='38422']
Hi,
I have a question about that last comment - so you are saying, its important to clean up the cDNA using spin columns or another method.
But its very difficult to quantify cDNA.
Did I get that right?
Which spin column method do you use?
thanks
Do you mean that I should to precipitate my cDNA reaction before to proceed to Real Time PCR ?
[/quote]
[/quote]
It is important to clean up the cDNA if you want to quantify it. However if you are doing simple RT-PCR then cleaning up is unnecessary
It is also important to clean up your cDNA following 2nd strand synthesis when proceeding to in vitro transcription
I use either the ChargeSwitch PCR clean up kit or RNA kit to clean up my cDNA, depending on whether it needs to be RNAse free or not
[/quote]
Do you realy manage to get rid of all RNAs? 
If, not - is there really any point in quantifying the cDNA?
[quote name='Gerd' date='Jan 26 2006, 11:19 AM' post='38648']
[quote name='John Buckels' date='Jan 25 2006, 02:33 AM' post='38510']
[quote name='smurray' date='Jan 24 2006, 11:08 PM' post='38422']
Hi,
I have a question about that last comment - so you are saying, its important to clean up the cDNA using spin columns or another method.
But its very difficult to quantify cDNA.
Did I get that right?
Which spin column method do you use?
thanks
Do you mean that I should to precipitate my cDNA reaction before to proceed to Real Time PCR ?
[/quote]
[/quote]
It is important to clean up the cDNA if you want to quantify it. However if you are doing simple RT-PCR then cleaning up is unnecessary
It is also important to clean up your cDNA following 2nd strand synthesis when proceeding to in vitro transcription
I use either the ChargeSwitch PCR clean up kit or RNA kit to clean up my cDNA, depending on whether it needs to be RNAse free or not
[/quote]
Do you realy manage to get rid of all RNAs? If, not - is there really any point in quantifying the cDNA?
[/quote]
The answer to your quaestion Gerd is no, and no
I normalise by total RNA input
me, too, John; it's the only way to control the template amount, there are too many variables otherwise
Don't you guys construct a standard curve using a plasmid and then use a relative standard (like Beta actin or GAPDH) for quantitation?
That seems like the only meaningful way to do it.
-Matt
me, too, John; it's the only way to control the template amount, there are too many variables otherwise
Don't you guys construct a standard curve using a plasmid and then use a relative standard (like Beta actin or GAPDH) for quantitation?
That seems like the only meaningful way to do it.
-Matt
I usually serially dilute an appropriate RNA control to make a standard curve prior to first strand synthesis. Either that or serially dilute cDNA made from a normalised input of RNA
What do you mean by a 'normalised' input?
Do you mean that you measure using the spectro and dilute all RNA samples to one standard conc, then assume RT efficiency is the same for everything, then dilute the resulting cDNA based on the standard conc of RNA?
Sorry, I'm a little confused
thanks!
I usually serially dilute an appropriate RNA control to make a standard curve prior to first strand synthesis. Either that or serially dilute cDNA made from a normalised input of RNA
What do you mean by a 'normalised' input?
Do you mean that you measure using the spectro and dilute all RNA samples to one standard conc, then assume RT efficiency is the same for everything, then dilute the resulting cDNA based on the standard conc of RNA?
Sorry, I'm a little confused
thanks!
My "usual" method goes along these lines:
Quantify RNA by OD absorbance
Dilute RNA to 100ng/ul
Use 5ul (500ng) RNA per RT reaction
Assume RT efficiency is the same, which it should be providing the RNA is of equivalent purity
After RT, dilute all samples 1:5 with water
I have a control cDNA sample generated in the same way which I dilute serially 1:2 to generate my standard curve. However I am not doing any stringent analysis of expression, just checking general mRNA levels are similar throughout my pools of samples
I understand now.
cheers!
I disagree with your assumption that the RT efficiencies should be the "same."
I find that is seldom the case (they can easily vary by 10-20%), which is why I use a diluted plasmid of known concentration (it's easier to accurately OD a plasmid anyway) for plotting my standard curve and a relative, constituitively expressed internal standard to compare between samples. Ideally you'd purchase an alien RNA sample of known concentration to spike your sample before RT, but that is generally not possible.
My method involves more cloning, but it also produces much better results, I find.
-Matt