6 replies to this topic

[Abiotic_Kabir]

Hi,
I have designed primer using the mRNA sequence. I also got bio-rad cDNA synthesis kit. I am wondering whether OligodT or random hexamer will be convenient to use for first-stand cDNA synthesis? Thanks.

Cheers!

[pcrman]

You can use either type of primers and you should know the difference between them and be able make a choice based on the purpose of you experiment.

[Trof]

Oligo dT primers only transcribe eukaryotic mRNA that has poly A tail. Only mRNA fraction in your RT reaction would be transcribed, but 5' ends of mRNA may be less abundant, depending on the integrity of your RNA.

Random hexamers would transcribe everything including rRNA, which makes the bigger fraction of total RNA, usually you don't need it. But if you're going to use 18S for normalisation, you can't use solely oligo dT primers.

We use the combination, put both primers into reaction. So the mRNA 3' is enriched, but polyA-missing fragments are also transcribed.

[Abiotic_Kabir]

thanks for the replies. Yes, I am working on eukaryotic plant gene. I didn't get any band by using oligo dt even for the actin (housekeeping gene). I designed the primers using mRNA sequence by the help of primer3 software though. I am really confused what's wrong   You guys have any suggestions?

Kabi

[Trof]

You either didn't get cDNA well or your PCR primers (PCR reaction) doesn't work. You must test which hypotesis is right. So get a working cDNA from the same species or get a working pair of housekeeping primers (with optimized conditions) to test on your cDNA.

(and just for the record, I'm not a guy )

[Abiotic_Kabir]

Hi Trof,
When you put both oligodT and hexamer in the same tube, what PCR program (temperature cycle) do you use?

[Trof]

You mean what RT temperatures we use (since RT isn't technically PCR, it doesn't cycle)? Recomended by the kit, (Transcriptor from Roche) same as for random primers only, but this may depend on the RT enzyme you use, use their recomended temperatures for random primers.

In our protocol:
Up to 4 kb RNA length - 10 min/25°C then 30 min/55°C
more than 4 Kb - 10 min/25°C then 60 min/50°C