Re-amplify PCR product by realtime PCR? - (Jul/26/2006 )

Hey guys,

Do ya'll think its posible to use PCR products to run real time PCR?
SYBR Green.
Coz the concentration of my GOI is really really little even after normal PCR. ONly a faint band is produced. And I can't do a dilution curve for my GOI as even 5X dilution can't amplify in realtime.

For relative quantitation using Pfaffl method where only the PCR efficiency of a standard curve and CT values of samples are needed. What if :

1. I used PCR products of my HKG and GOI to do their respective standard curves in realtime

2. And then use PCR products of control and treated samples to compare their CT values.

Thanks for your inputs...much appreciated.....

Chris

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The normal PCR works as a real-time: i.e. you have a logaritmic phase of amplification and then a plateau, therefore you should be really careful not to be in the plateau phase when you get your sample for real-time. If you do a real-time is because you want to do a comparison of the expression levels of the target genes. Do you think it is possible to compare something that has been already amplified? Would it be accurate? I don't think we would need a real-time if it were. Moreover, cannot you start from a higher amount of sample? This would solve your problems.
In conclusion, I don't think it's a good idea.

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I agree

you cannot compare relative expression if you are starting from material that essentially has no control over amount. your data would be meaningless, and I suspect un-repeatable

sorry!

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thanks for feedback...

So...how do I increase the concentration or amount of my cDNA? I prefer to spectro cDNA, not RNA because I believe RNA consists of different kinds of RNA but only mRNA converts to cDNA. right now I always make 20ul of cDNA from from 9ul RNA. Can I use ethanol precipitation to increase cDNA yield? Please help me. Realtime is killing me and I'm nearly at the end of my wits...

Chris

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Cannot you use more cells, bacteria, tissue or whatever you use to get your RNA?

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Hi DNAfactory...

Coz I measure cell density of OD 0.8 at 600nm. I'm using Qiagen RNeasy Minikit, and I think there is only so much cells I can use until the membrane clogs up...BTW I'm using Candida albicans cells.

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How many ul do you use to elute? Do you elute twice? Maybe you can use 2 samples and two columns for each RNA extraction and in the last step you can use the eluate from one column to elute the RNA from the other as well. In this way you should be able to have double the concentration in the same volume. Cannot you otherwise change the extraction method? You can use the midikit, for example, that allows you to get 10 times more RNA than the minikit

http://www1.qiagen.com/Products/RnaStabili...RNeasyMidi.aspx

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Hi Chris,



Check this out: https://products.appliedbiosystems.com/ab/e...&tab=DetailInfo



Just came across it today.

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Thanks everyone for your idea.

Dear DNA.
I think the volume of cDNA in realtime is a limit. And even if I get lots of RNA my RT product is quite limited. But maybe I'll try the 2 column thingy...

Dear soluene,
Taq is expensive, and I have 3 GOIs;I don't think my boss wants to pay so much $$$

How about if I do a standard curve using purified PCR product to include the range of the expected GOI and then compare control and treated using cDNA? DO you think its possible? If I can't get a solution, I think I'll juz quit realtime.

Chris

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I am not saying this is the only way, but this is the way I do it and it works quite well for me:

1. I prep the RNA using the tricks to increase yield, suggested by manufacturer
2. I elute in a large volume and ETOH/oAC ppt, resuspending final pellet in a small volume, to increase concentration. I find that I get more recovery, then if I elute in a small volume in the first place; I think some of the RNA stays on the matrix if you don't elute with a sufficient volume
3. I spec the RNA and put a defined amount into each RT, using oligodt primers; I put an aliquot into the qPCR reaction

I think it is better to spec your RNA than your cDNA


"And even if I get lots of RNA my RT product is quite limited"
I think perhaps you need to look at this step? what are some details to your method for performing the RT?

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