3 replies to this topic

[raves]

I need to increase the amount of RNA i am getting. I have a number of aliquots of recently extracted rna (approx 200ng of RNA per tube (20ul volume)). I need to be able to get approx 5ug of RNA in total for the next experiment.This means i would need to perform a further 25 extractions and combine all the aliquots together to get approx 5ug in total for sequencing.
If anyone had any other ideas as to how to improve total amount of RNA. In combining all the aliquots together i wont lose any RNA will i?, but i could change the quality (I check all the RNA on an agilent PICO chip (RINs are between 5.7-6.9).

Could i post cleanup this entire pool that i make? Is there a good protocol for this. Again would i lose RNA?


thnx

[jah]

How are you preparing the RNA....RNeasy, Trizol, other?  and from what source...tissues, cells, plants, soil etc.  Need more info to be of much help

[raves]

jah, on Jan 4 2010, 06:53 PM, said:

How are you preparing the RNA....RNeasy, Trizol, other?  and from what source...tissues, cells, plants, soil etc.  Need more info to be of much help

Im using an rneasy plus mini column to extract rna from my sorted cells.I only get approx 200ng rna from my sorted cells. I need to get around 1-5ug for sequenicng. Is it best to combine the rna aliquots asnd then clean this up or combine the sorted cells and do one big rna extraction

[gogreen]

I guess it would be better to pool the cells and then do one isolation than to do a lot of them with less number of cells...How many cells do you use per extraction? is your yield correlating with the expected?