single stranded cDNA storage

[susi]

Hi everyone!
I am about to start an experiment where I'm going to do a real-time PCR with cDNA as template (real-time RT-PCR). I will start with extracting RNA from soil and do a first strand RT-reaktion. What is the best temperature for storing single stranded cDNA? For how long will it be stable at this temperature and give good results in my real-time RT-PCR?

Susi

[LilKim]

When you extract RNA (some if it will be single, or double stranded). Of course you're only interested in the single stranded RNA (particularly mRNA) for you RT rxn. Keep the RNA FROZEN -80 and let it thaw on ICE and keep it on ICE if it's out of the fridge ... because I can be degraded very easily.

After your RT rxn you'll have double stranded cDNA... since DNA is more stable than DNA you can keep it at 4 degrees (for short periods of time... like a few weeks). And keep it at -20 for extended periods of storage.

good luck!
- kim

[susi]

I'm planning on just doing the first strand reaction (skipping the second strand reaction). Will single stranded cDNA be as stable as double stranded cDNA?

[LilKim]

maybe i'm going senile... but, i'm not sure why you'd want to skip the second strand reaction (or should i say.. what are you trying to do?)

please enlighten me?

thanks!
- KIM

[susi]

I am going to use the cDNA directly in a real-time PCR reaction and was told that it's not necessary to make doble stranded cDNA for this purpose.

We are going soil-sampling next week, and will return with a lot of samples. I want to quantify many different genes from the same
RNA --> cDNA-isolate, but I'm not sure how to store (short term or long term) the cDNA aliquotes until use in the different PCR-reactions.

Susi

[oppox]

Cant u ask the ones who told you what to do. Singlestranded should be more unstable than double, I dont think storing it at -80 will hurt.

[RompingRibozyme]

Dear Susi,

The RNA you extract is single stranded with the exception of small RNA which have self complimented and have been cleaved as we see with RNAi. When you perform an RT-PCR, you are using the ssRNA as a template and your resulting amplicon is a ssDNA known as cDNA. cDNA is always single stranded, with the exception of a DNA:RNA hybrid complex which may form from the annealing of the ssDNA with the ssRNA. I assume you now want to quantify your transcript levels, this is done by qPCR (quantitative PCR) which is done after your RT-PCR.

DNA is stable at 4 degrees C. For long time storage I would store it at -20 degrees C in a 0.5x-1.0 x TE. I prefer 0.5X TE since that won't interfere as much with any down stream reactions you wish to run.

Cordially,
Peter