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minimum amount of cDNA? - molecular biology (Nov/26/2009 )

hi,
i have extracted 90 samples of total RNA from 9ml of whole blood each, using TRI reagent. the concentration of RNA that i got from them is on an average 600ng/ul or you can say 0.6ug/ul. i need to process these samples for 2 step real time RT-pcr. i converted 2.5 to 3ul on an average i.e. (1.5ug) of this total RNA to cDNA using high capacity cDNA kit (ABi) in a 20ul reaction.

i will have to dilute it 1 in 3 parts for getting sufficient volume amount for running the same in duplicates of each, 1st my gene of interest and 2nd a house keeping gene, so total 4 reaction of 20ul per sample.

therefore 1500/4=375ng per 1vial of 20ul reaction

now the question is that only 375ng or 0.375ug of total RNA converted to cDNA is present in each vial of 20ul reaction, is it sufficient enough for real time pcr? the instrument that i use is a ABI 7500. the primer sets are an optimised assays from ABi and so is the cDNA prep kit.

note: i am talking in terms of RNA because i read here that its not advisable to quantify cDNA since dNTPs primers etc may still be present and give wrong readings..

please help me out, and let me know if i have missed out on anything in writing or doing.

-SRM MRS

-srm mrs-

I would even say that this is far too much input material, even for convential PCR this is pretty much. Typical amounts for real time PCR are ranging from 5 to 20 ng of cDNA. Putting too much cDNA into the reaction is 1) a waste of material and 2) it increases the possibility of PCR inhibition by coisolated impurities or salts from the RT reaction. Especially with RNA from blood I would be very cautious as heme is a potent PCR inhibitor. I would recommend to do first a serial dilution curve with your cDNA starting with 100ng down to eg. 0.001ng, depends on how strong your target gene is expressed. Then you will see if you have any inhibition effect.
With input amounts >100ng I don't get any amplification in 10Ál volumes and I'm also using the ABI high capacity kit.

-tea-test-

tea-test on Nov 28 2009, 12:09 AM said:

I would even say that this is far too much input material, even for convential PCR this is pretty much. Typical amounts for real time PCR are ranging from 5 to 20 ng of cDNA. Putting too much cDNA into the reaction is 1) a waste of material and 2) it increases the possibility of PCR inhibition by coisolated impurities or salts from the RT reaction. Especially with RNA from blood I would be very cautious as heme is a potent PCR inhibitor. I would recommend to do first a serial dilution curve with your cDNA starting with 100ng down to eg. 0.001ng, depends on how strong your target gene is expressed. Then you will see if you have any inhibition effect.
With input amounts >100ng I don't get any amplification in 10Ál volumes and I'm also using the ABI high capacity kit.


hi Tea-test,
Thankyou very much for a prompt reply,
this solve major part of my query..... but something still bugging me is that, isnt mRNA just about 5 to 10 percent of total RNA, will it be still sufficient? and one more thing you had mentioned 5 to 20ng of cDNA..... i did not quantify the cDNA..i quantified the RNA and based on this i mentioned the ug quantities, so how can i be sure what is the concentration of cDNA?
but once again thank you very much!!!

-srm mrs-

hi,

with ng of cDNA I actually mean the RNA equivalents so we are talking about the same thing.
and yes, usually this is enough input material as real time PCR is an extremely sensitive method (down to one molecule in your sample). but I would recommend you to do first a serial dilution series with a high expressed housekeeping gene like B-actin to rule out any possible inhibition effects.

-tea-test-

tea-test on Nov 29 2009, 09:10 PM said:

hi,

with ng of cDNA I actually mean the RNA equivalents so we are talking about the same thing.
and yes, usually this is enough input material as real time PCR is an extremely sensitive method (down to one molecule in your sample). but I would recommend you to do first a serial dilution series with a high expressed housekeeping gene like B-actin to rule out any possible inhibition effects.



hi,
thank you very much, and i am using beta actin itself as my housekeeping gene(hkg). i was plannning to make serial dilutions of my positive control for both my Gene of Interest(GoI) as well as beta actin.. but i was just waiting to know if those amounts were enough...now that i know they are enough ( thanks to u) i am going to carry out the experiment in 2 slots pretty soon... 1st for the serial dilutions and then later all my 90 samples run in duplicates for each GOI & HKG.. Wish me luck!

-srm mrs-

hi,
i tried out my pcr reactions but i got some unexpected results...
i had normalised all the RNA samples before i converted them to cDNA as you suggested.... i got them all to ~385ng per reaction. the cDNA was also checked on agarose gel,, and they all gave very similar smear bands... almost equal in density.

but when i carried out two samples for trial for real time pcr, the following are the results i got.

Gene of Interest (abca1)
Samples in duplicates / CT values / Mean

A1 healthy normal / 19.42 / 19.34
A2 healthy normal / 19.26

B1 patient sample / 22.49 / 22.43
B2 patient sample / 22.37

C1 NTC / Undetermined
C2 NTC / Undetermined



House keeping Gene (beta actin)
Samples in duplicates / CT values / Mean CT

A1 healthy normal / 20.59 / 20.62
A2 healthy normal / 20.65

B1 patient sample / 16.61 / 16.65
B2 patient sample / 16.69

C1 NTC / Undetermined
C2 NTC / Undetermined


now here i got two values for beta actin with quite a big differrence
how can i interpret the results? can i normalise the beta actin values with such a big difference? The patients will have a later CT value since the expression of abca1 is expected to be lower in patients with lower HDL values... which came out as expected. but what i am worried about is the beta actin values...since every cDNA with same ng quantity should give similar CT values for beta actin irrespective whether its a normal or a patient sample, right??

plz help.

-srm mrs-

it seems that b-actin is somehow induced in your disease state. Are you sure that you have loaded the same cDNA amounts? Have you done the dilution curve? what was the dilution range and what was the slope of the curve? what was the input amount of cDNA/reaction in your samples? Can you check some other HKGs like GAPDH, UBC, HPRT,...? There is the possibility that b-actin is not a suitable reference gene for your specific experimental approach.

-tea-test-