Are you doing this for quantitation or just to detect the presence of differentially spliced products?What size RT-PCR product are you intending looking at - 4kb? I would think it likely that the randomn primers will prime several times within the 4kb. Oligo dt will give you the "longest cDNA, but a 4kb transcript will depend on how good your starting template is. If you are just intending to look at difference between a 200 and 400 bp PCR product you can get away with randomn priming. A hexamer will bind on average once every 4096 bp ( assuming 100% identitity - in practice you may get partial priming as well)).You may have to do 1h extension - look up the processivity of the Superscript 3. Have you also considered a target specific primer downstream of your splice sites?

ian

Hi Ian,

Thanks much for the reply!

I'd be looking to detect presence of differentially spliced products first, subsequently work on to quantification based on physiological changes. There is a total of 8 splice variants I'm looking at.

My RT-PCR products would range between 140 to 460+nt. The primers closest to the 5' end sits starting ~82nt of the mRNA.

Processivity of the Superscript 3 is about 40nt per binding event according to Life Technologies fact sheet. Yes, I have considered target specific primers; would probably have to work towards it as an alternative to oligo dTs and random hexamers.. The only matter now is to design something that would encompass all the variants.

Much appreciation for your input!

JJ

Are you looking at a single target with multiple splicing eg a cloned EBV virus with alternative splicing, or mutiple targets with multiple alternative splicing (eg a collection of patient samples with a variety of different possible splicing)?You may have to consider a" concensus" priming oligo for RT and splice specific oligos for amplifying the individual splice varients. Bear in mind you must be able to discrimminate between the mRNA and genomic DNA when designing primers and performing amplification - Incorporate DNAse digestion, primers across splice junctions etc

Ian

I am looking at a single target with multiple splice variants.

Noted on the DNA contamination; have used DNase during sample collection!

JJ

I'm doing a similar type of experiment. I had been using superscript II, but switched to Roche's Transcriptor system. It is much cheaper than the superscript III (similar in price to superscript II kits) and it uses anchored oligodT's which helps to ensure the reverse transcription begins at the start of the polyA tail of mRNA (which can be very long and reduce the amount of mRNA transcript RT'ed). They indicate special conditions for priming sequences over 4kb and although some of mine are right about that size, I have had very good results using it.

For my experiment, I designed my primers so that 1 of the set actually straddles the splice site of interest (over the exon boundary of the splice site). This helps to eliminate DNA confounds, but more importantly keeps my reactions specific to 1 product (only 1 splice variant) and allows me to ensure that the product is ~100-150 bp which is ideal for real-time PCR. If you plan to use 1 set of primers for multiple splice variants in real-time PCR, the efficiency of the reaction will be greatly influenced by the size of the product and the number of products being made. It takes more work initially to design a set of primers for each splice variant, but it makes analysis much easier and more uniform. Plus, Primer3, the free web-based primer design program, can design the primers to any point along the sequence.

Good Luck!