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

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QUOTE (aimikins @ Jul 29 2006, 01:01 AM)

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?

Thanks aimikins for your insight.

I meant that my GOI doesn't amplify well. SAy I get 1 RNA means I get 1 cDNA. So no matter how much RNA I produce, there is only so much cDNA I can produce ( I think the mRNA expression is quite little). Because realtime recommends only putting 10%of total volume of RT reaction, I can't put too much cDNA. The RT protocol is as below.

1. 5ul H2O+ 1ul specific primer+5ul RNA
2. 70C 5 min
3. ice bath 3 min
4. spin down, +8ul master mix+ 1ul MMLV (mastermix = 4ul 5X reaction buffer+2ul 10mM dNTP mix+1ul RNasin+1ul H2O)
5. incubate 37C 1 hour
6. 70C 10 min
7. ice bath 2 min
*all from Promega

I think I should change the incubation time to follow the specific primer's. I used the protocol for random hexamers and subsituted with specific primer. I found it produced better CTs.

Thanks...

Chris

-chris_sylim02-

Hi Chris,

yes, its definitely possible to use PCR product to make your standard curves and then use cDNA to do the assays.
I have done this and my results were good, even though my gene is expressed at very low levels (Ct values are 30-37), which would have made it nearly impossible to make a standard curve using cDNA.

-smurray-

By the way, is this the same gene that you showed in your other thread, where your E values were low? This might also be a reason for the late amplification. Can you redesign your primers?

-smurray-

QUOTE (smurray @ Jul 31 2006, 12:29 PM)

By the way, is this the same gene that you showed in your other thread, where your E values were low? This might also be a reason for the late amplification. Can you redesign your primers?

Hi Murray,

So good to hear someone agreeing with me. Now I have some confidence that someone has done it before. Yes its the same gene. and another one like it. also low expression levels.

Nope I would like to retain the primers. I would have changed them earlier if I didn't want to keep them. The reason is i had done semi-quantitative reverse transcription PCR using those primers and now I have to validate with realtime. So I have to retain the same primers.

Thanks so much for everyone's ideas. SO now this is what I'll try:
1. use 2 column to purify RNA, and eluting from one column to another twice like aimikins said.
2. add more RNA into my RT reaction, maybe doing a 50ul reaction.
3. use the specific temperature for my specific primers during RT incubation.
4. use PCR products to plot a standard curve like murray said.

I'll give it a shot and see what happens.
Thanks everyone!!!

-chris_sylim02-

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