plasmid stnds linearized, cDNA clean-up...low copy genes..

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plasmid stnds linearized, cDNA clean-up...low copy genes..

Postby Neke » Jan 28 2005 7:59 pm

Will linearizing my plasmid standard lower its Ct values? I prepared my plasmid standards according to ABI info so that my standards go from 300,000 copies down to 30 copies. However the 300,000 standard Ct values are around 25. I thought this value should be much lower.
I'm doing 2 step RTpcr ( with Sybr green) to assess viral load. When looking for low copies of virus whats the maximum ug of RNA people use to make their cDNA. I thought that if i maxed out the RNA amount (16ug with SSIII, using oligo dT) in my RT reaction then i would for sure catch the low viral loads. Now i'm not sure if this is the right logic. I'm also wondering if anyone cleans-up their cDNA to get rid of inhibitors?
Thanks in advance for any helpful information. :)
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Couple of suggestions for you

Postby Suzanne » Jan 29 2005 12:18 pm

Hi Neke,

I am not sure about the answer to your question on viral load, but regarding linearization of plasmids, I can direct you to this post (you've probably already seen it by now):
http://%20molecularbiology.forums.biotechniques.com/forums/viewtopic.php?t=10124

Apparently it is important to linearize plasmids for the lowest Ct value. However, you can always do a side by side comparison yourself and see if for your gene, you see a difference.

To maintain sensitive detection in samples, many people use a carrier RNA in the stored cDNA to help prevent loss of detection due to stickiness of the cDNA to the sides of the tube. We use 10ng/ul and some people use 30 ng/ul:

http://%20molecularbiology.forums.biotechniques.com/forums/viewtopic.php?t=10157

This seems to help allow for detection down to low copy numbers. You can use tRNA or homopolymer A.

Regarding the amount of RNA to use, I think this really depends on how much you have to start with. Many people doing viral load testing on blood, serum, and plasma will have ng-pg quantities to work with and still be able to sensitively detect virus. Sometimes people will have to do a nested PCR to get the product to a detectable level. Since it sounds like you have a lot of RNA to work with, I think you have an advantage. Why not try priming RT with a gene specific primer for the virus instead of RTing the entire mix with random primers or polydT?

Hopefully someone with direct viral load testing experience can give you more specific insight.

Best,
Suzanne
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