if the primers stopped working when you changed to a new RT kit then I would change back to the original kit, Do you have a colleague who makes cDNA that you could test your primers against just to test if the kit is simply not working as I expect primers to be stable,Check that nobody has changed your pcr settings but the kit has many components so more likely to go wrong than primers. Are your housekeeping genes amplifying out of your cDNA kit product? or does nothing amplify.

if the primers stopped working when you changed to a new RT kit then I would change back to the original kit, Do you have a colleague who makes cDNA that you could test your primers against just to test if the kit is simply not working as I expect primers to be stable,Check that nobody has changed your pcr settings but the kit has many components so more likely to go wrong than primers. Are your housekeeping genes amplifying out of your cDNA kit product? or does nothing amplify.

Paul Rutland Thank you. I managed to generate cDNA from three different kits and test them with qPCR for the amplification of my neonatal gene. Two other kits (other than mine), actually provide successful amplification. So, I went back and check the length of my neonatal gene mRNA and it is quite long (8000bp). So, with my qPCR primers (for the neonatal gene), it amplifies a site near the 5' ends of the mRNA. I compare it with other genes that can be amplified by the cDNA from my kit and none of them are that long or have a target site near the 5' end. This shows that maybe 3' bias are happening, where the oligo dt primers did not manage to allow perfect synthesis of cDNA from the polyA tail until the 5' ends due to the mRNA is long. This is proven from one of the kit that successfully synthesized my neonatal gene cDNA, where I only use random hexamer and no oligo dt. My kit and the other kits seems to provide combination of both random hexamer and oligo dt. So, the synthesis of my neonatal gene cDNA is much dependent on random hexamers. So, the real problem is the protocol provided by my kit. I need to incorporate steps that allow better priming of random hexamers (since the kit have both random hexamers and oligo dt) or increase efficiency of RT activity by prolong the incubation time or increase temperature since it is suggested that higher temperature are better for complex RNA (perhaps it refrains RNA from having secondary structures while synthesizing the strand). I consider my mRNA as complex due to it being long and prone to have secondary structures. Basically, as you said, my primers are all good and stable. It is indeed the RT kit itself (the protocols, actually). Thanks!

thank you for the update Ahmad. If you want better rando priming try using random 9 mers. Very cheap if you have it made as an oligo and not bought off the shelf or you could try using gene specific primers to amplify the rna

best wishes

Paul

Agree with Paul Rutland.