difficulty synthesising cDNA from total RNA - (Apr/09/2008 )

Hi everybody,

I'm having difficulty synthesising cDNA from total RNA. Here's what I've done:

1. Trizol extraction to extract total RNA
2. Verified RNA integrity on gel.....RNA is intact
3. DNase I (Invitrogen) treatment exactly as stated in the protocol (room temperature incubation for 15 minutes, inactivated by addition of 2.5mM EDTA and 65 degrees for 10 miutes)
4. Reverse transcription using Superscript III Reverse Transcriptase (Invitrogen)........used oligodTV primer, 50 degrees for 1hour, heat inactivated 70 degrees 15 minutes)
5. RNase H (Invitrogen)treament 37 degrees 20 minutes
6. Column purified the final product to obtain DNA only.
7. PCR.........NO PRODUCT SEEN ON GEL

Note: Primers and PCR conditions tested on genomic DNA and it works perfectly.
DNase I treatment is a MUST in my case.

I'm assuming that either the cDNA synthesis isn't working at all or DNase I treatment has degraded my RNA. I must stress that I MUST MUST MUST have DNase treament. Please advise. Thanks.

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Have you run some of your RNA on a gel after DNAse treatment instead of before? That way, you could at least verify that you're starting with good RNA for your RT reaction. (And hopefully rule out the DNAse treatment as the problem)
Are you doing an alcohol pptn after DNAse?
Are you also running PCR with primers for a housekeeping gene as a positive control?

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Thanks for the reply.
I didn't run the RNA after DNase treament. However, I've tried reverse transcription on RNA WITHOUT DNase treatment and it still only gives 1 PCR product corresponding to the genomic sequence.

No, did not do alcohol precipitation. How does it help?

Housekeeping gene? not yet. I will be doing it with the next batch of cDNA I attempt to make. I'm planning to use GAPDH as my housekeeping gene.

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Do you spec your RNA after isolation? How much RNA are you using in your RT reaction?
Do you need to column purify? I have never done this. Are you using the correct kit for purification? Many have size limits.

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Hi.

Yes, I nanodrop my RNA samples after extraction. A260/280 is 1.9 to 2.
I use 1ug total RNA in the RT reaction.
As for purification, I just use QIAquick PCR purification kit to remove any residual enzymes, dNTPs etc.... I don't think it's essential but some people I know do it just to clean up the sample. Their PCR after that works fine. The size shouldn't be a problem. The longest mRNA transcript is only 1.3kb.

Thanks

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We just had a similar problem. The solution was that so many people used the RT kit for making cDNA that the DTT was not working anymore...we never had cDNA (checked with actin primers)...solution: new Kit for RT...everything is ok now...

Maybe that helps...

Stardust

PS: I have never done the column purification...you loose a lot of material...maybe try without once?

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Is the gene you interested expressed at high level, if not (I mean it maybe a rare expressed gene), it maybe difficult to detect with a single PCR. Have you checked the cDNA by PCR-amplifing of some house keeping genes such as actin and tublin or even some other known rare expressed genes. If they works fine, it is not the cDNA synthesis problem, but your target are rare expressed and difficult to be amplified.

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I'll try switching to a fresh batch of DTT.
Will also try without the purification.
I'm not sure if this is might be the problem: When I resuspended my RNA pellet after RNA extraction, I did it in 0.5% SDS. Then I store it at -80. When I thaw it to aliquot some for the RT reaction I tend to briefly heat it (15 seconds roughly) at 55 degrees which helps aliquoting easier. Perhaps I degraded the RNA by doing that??? I'm assuming it won't affect it much since all the steps (DNase and reverse transcription) up until the final product includes numerous heating procedures.

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One of the genes I'm looking for is expressed highly in the tissue I'm investigating. Will try the housekeeping gene as soon as possible.

P.S : Your english is fine

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Do your primers span an intron? (This will help rule out DNA contamination when you do get a PCR product).
An alcohol precipitation is usually done after DNase treatment to get rid of salts, enzyme, and free nucleotides, all of which can mess with your RT reaction.
A housekeeping gene is very important as a control, and will tell you 1) that your PCR works and 2) that your RNA is good.
Just make sure that the fragment you amplify of your hk gene is different enough in size from your target gene so you can see them both in your ++ reactions. Also make sure to run other reactions with your hk and target genes seperately though, in case the primers interact or if competition favors one product over the other...

You can use up to 5µg RNA in your RT reactions -- maybe you need to increase yours a little.

Also, you really shouldn't need to column purify your PCR product before running on a gel. You shouldn't see anything other than your DNA anyway, and adding extra steps just adds more chances to lose your product...

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