cDNA for qPCR - Help with basics please (Mar/08/2010 )

Hello,

Am new to molecualr biology and am currently struggling so any help would be much appreciated. I want to make cDNA from pre-extracted RNA from plant material. I am then hoping to carry out qPCR with the samples and comapre the expression of a particular gene. All of which I have never done before. So I have some very basic questions:

1.Before I start with the cDNA process should I use DNase to remove genomic DNA from my samples? I have read that this is important. If so is there a kit that is routinely used to do this (I am looking to do things as cheaply as possible).

2.RNase inhibitors: I have limited RNA extracts should I use RNAase inhibitors to help protect my samples? If so again can any one recommend a specific treatment which I can use when making cDNA.

3. I have inherited a cDNA synthesis kit from my predecessor (Roche Transcriptor High fidelity cDNA synthesis kit). Here there are two choices of primers (random hexamer primer and Anchored-oligo primer (dT)18 primer). Am not sure I fully understand why two sets are included and which set I should use (I know one targets all RNA, the other mRNA??)

4. Once I have managed to make cDNA will be carrying out qPCR on a Roche light cycler to compare gene expression in my samples (primers have previously be designed for this). Does this sound reasonable?

Any help/protocols you can offer would be great, am really struggling at the moment!

phosphate girl on Mar 8 2010, 07:27 AM said:

Hi,

I am new to rt/q PCR and found a couple of articles that may help with basic questions, that are easy on the brain cells when you're just starting. I'm attaching a trouble shooting guide that was posted by another member too. Hope they get you started in the right direction.

Denny on Mar 8 2010, 06:31 PM said:

I just googled "plant cDNA protocol" and found a ton of links, here's one.

http://www.protocol-online.org/prot/Molecu...-PCR/index.html

Thanks guys, your help is much appreciated. I was also wondering if either of your knew of the best way to store cDNA. I was thinking in nuclease free water or TE buffer (pH 7.0) at -20. Do you think this would be OK?

Thanks again for your help in demistifying molecular biology!

phosphate girl on Mar 8 2010, 04:27 AM said:

1. Absolutely not necessary if you use Taqman probes which are designed across intron/exon boundaries. Also check RNA on a gel (I like glyoxal for RNA) and see what you have. If you use SYBR for your real-time though it is advisable.

2. No, as long as you are careful and use good technique you dont need them. Don't listen to most of the grad students on here who have been brainwashed into thinking this. Don't mask poor technique and practices with inhibitors, work on your technique and be meticulous.

3. The only thing you need to worry about is that one is usually better for longer, full length ORF cDNA transcripts (18 mer/oligo dT) and one is better for shorter fragments (random hexamer). Basically, the oligo dT will bind to the poly-A tail of the mRNA and read off from there creating, in theory, more full length open reading frames. The hexamers will bind randomly and will usually give much shorter fragments in random places througout the gene transcript. In my experience it does not really matter for qRT-PCR because you are dealing with very short transcripts (<200 bp usually). You could use either and I would probably use the hexamers. Now if you were going to be doing longer PCR after cDNA (i.e. cloning a ORF), you would need to give it more thought and I would recommend oligo dT for this all day.

4. Yes of course. Make sure you normalize the amount of RNA going into the cDNA reaction (i.e. 0.5 ug of each sample) and then load equal volume of cDNA into the real-time. Quantifying cDNA is a little tricky so its easier to quantify at the RNA step.

Hope this helps

Dukey on Mar 10 2010, 07:46 PM said:

Hello Dukey,

Yes thank you so much that does help a lot and I was wondering about whether to normalise the RNA or cDNA concentration so thankyou. Am guessing the best way to get similar quantities or RNA between samples is to measure their concentration individually using OD (we have a nanodrop) and than dilute as appropriate with pure water or buffer? I was going to run a 1 % agarose gel to check the quality of my RNA extracts...am not sure what glyoxal is? Do you think agarose would be OK. Lastly somebody suggested I check the purity of my RNA extracts using the nanodrop (A260/A280 ratio). This seems sensible and if the purity is poor am I right in thinking I can clean-up the extracts with a kit?

Sorry if these questions seem a little obvious, I just don't want to make any massive mistakes first time round.

its ok to use a normal agarose gel to check for the RNA quality, I'm always doing it like this and it's also a good idea to change the running buffer before because I have seen that old running buffer can degrade your RNA.