Hi, Qiang, at first, you can use normal PCR, cloning and sequencing to identify all of the splicing variants, then you can design specific primers to check the abundance of each. Hope this help.

I would suggest, design specific probes for each predicted alternative spliced transcript and run northern blots. Once you have got the right length on the blots you could run specific PCR for each transcript. If you want to clone those, do a "Rapid Amplification of cDNA Ends (RACE) cloning.

If there're reports of the 4 isoforms, make sure under what condition they identified them, for example, in what developmental stage, or under specific stimulation... Some gene isoforms only express in specific conditions. If the 2 isoforms are reported in your study condition, one other possibility is one isoform has very low expression, under this condition, you can just increase your PCR cycle # to 40, to see if you have a strong band and another weaker band. If these simple ways not working, as Saravana and Claire suggested, you can use RACE and probe to detect the specific one. Let us know if it works. Good luck

Hi Qiang,

you could try amplifying the full length cDNA obtained from cyclohiximide treated and non treated cells, of your gene. Then you could sequence multiple amplicon clones; gel band isolations may help increase the chances of obtaining multiple isoforms. Alternatively, you  could prep the sample and high throughput sequence barcoded libraries. Once you have the reads aligned to yor reference (make sure to use a splicing-aware mapper) you can use the Sashimi plot pluging in IGV to investigate the abundance of the different isoforms.

Good luck,

Najim