optimization of cDNA template for real time PCR array - (Dec/15/2006 )

Hi frnds!
I have to do some work on real time PCR arrays ie I would be going for gene expression profiling
of 84 genes which are expressed under a particular condition. Iam using super array kit for this, they have already dispensed primers in a 96 well format. I have to start from total RNA & there by it's conversion to cDNA, which will act as template for real time PCR reaction.some of the wells have primers for house keeping genes , into one well a non reverse trascribed total RNA is to be added & still into one other well no template is added.This was the whole story.
now my problems are:
1. how u all do your real time experiments, after converting RNA to cDNA do u all proceed on the same day for real time, or cDNA can be safely kept in alc after precipitating it with sodium acetate.
let me know the exact procedure of safely keeping cDNA template for long time say 20 days.

2. how should I standardize the amount of cDNA template for array plate( super array recommends to go for reverse transcription by taking 0.5-1 microgm of total RNA) it is what they recommend, I want to get the thing which is feasible for my experiment.


hope to get gud suggestions !!
poo

Hi there Poonam,

About the first thing, I keep my cDNA in ddH2O rather than in alcohol. You can precipitate it overnight and keep it in alcohol, but I haven't tried keeping it longer than overnight. Its easy to centrifuge it, take off the alcohol, then dry the pellet briefly and dissolve it in H2O. It will keep for several months at -20 degrees in H2O.

About the second question, you do the RT, then you usually finish with around 20 ul or so of product. If you started with 1 ug of RNA, and have 20 ul, that means the final conc is .05 ug/ul = 50 ng/ul. Then you just dilute as you want and add the same amount to each well.

Good luck.



1. how u all do your real time experiments, after converting RNA to cDNA do u all proceed on the same day for real time, or cDNA can be safely kept in alc after precipitating it with sodium acetate.
let me know the exact procedure of safely keeping cDNA template for long time say 20 days.

2. how should I standardize the amount of cDNA template for array plate( super array recommends to go for reverse transcription by taking 0.5-1 microgm of total RNA) it is what they recommend, I want to get the thing which is feasible for my experiment.

Hello Poonam,

if u r planning to run std curve for ur genes of interest and housekeeping genes....prepare a 10-fold diln series like 100...10... 1... 0.1... (nano/picogram, whatever) and so on to give reliable and reproducible results.
prepare a good enough stock of cDNA (starting with 1ug of RNA for 20uL reaction)...that will last long...since u will b doing optimization and finally quantification of 84 genes...try using the same cDNA every time u run a PCR....so,
prepare cDNA in something like...a 40 uL or even higher volume of RT reaction...to give 50ng/uL conc of final cDNA....dilute this stock as required.
for ex.
for 40 uL reaction volume use 2ug RNA and so on for higher volumes.

i hav always diluted my cDNA in TE buffer and stored them at -20oC.

Goodluck