4 replies to this topic

[shadow]

Got 108 RNA samples to do RT and real-time PCR detection. So RT and minus RT will be 316 reactions. real-time PCR with internal control and target gene, triplicate replications will give 108*3*2=648 reactions. Also 648 reactions for minus RT. Any idea to do a quicker and faster test?

Edited by shadow, 10 March 2010 - 11:40 AM.

[bob1]

It may look like a lot now, but it really isn't - only 15 plates with all reactions included. You could probably get away with not doing triplicates for the no-RT reactions. Duplicates should be plenty there, unless you know your primers will pick up genomic sequence too.

[shadow]

bob1, on Mar 10 2010, 03:40 PM, said:

It may look like a lot now, but it really isn't - only 15 plates with all reactions included. You could probably get away with not doing triplicates for the no-RT reactions. Duplicates should be plenty there, unless you know your primers will pick up genomic sequence too.

Thanks a lot! That's exactly what I am considering. But I am still suspicious of the results. How to normalize and how to make them comparable. There definitely will be big variations for one specific sample, which ct value will be trusted? Do you guys use some software to do the analysis?

[warsel]

shadow, on Mar 10 2010, 08:36 PM, said:

bob1, on Mar 10 2010, 03:40 PM, said:

It may look like a lot now, but it really isn't - only 15 plates with all reactions included. You could probably get away with not doing triplicates for the no-RT reactions. Duplicates should be plenty there, unless you know your primers will pick up genomic sequence too.

Thanks a lot! That's exactly what I am considering. But I am still suspicious of the results. How to normalize and how to make them comparable. There definitely will be big variations for one specific sample, which ct value will be trusted? Do you guys use some software to do the analysis?

I'm repeating basically what bob said: it's not that much - 15 plates can easily be done in a week's time!
Alternatives like 384-well plates for PCR or liquid handlers might make it a little easier, if awailable.

[toejam]

another option could be multiplexing. it sounds good but it can be a considerable amount of time to standardise. and bob is right, it's not as bad as you think.