how do i increase my rna yield

[sam1]

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.

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

[CharonY]

In order to give suggestions for increasing yield per isolation I would have to know from which substrate you isolate RNA as well as the precise technique.

 

Pooling does not result in loss per se, though intermediate handling (e.g. freeze/thawing, contamination with RNAse, etc.) can degrade your sample over time. Also, depending on your downstream application concentration may be an issue. In that case one can use one of the concentration columns or precipitation techniques. In this case a bit loss is usually to be expected.

[sam1]

Hi there i am extractin rna from some cell sorted on a facs machine. They are extracted using the qiagen rneasy plus miniextraction kit.each extraction results in approx total amount of 200ng RNA. I need 5ug for a downstream sequencing.

[CharonY]

That kit should give a higher yield (in the µg range). It sounds like there is an upstream problem. What cells are those and how much do you use per extraction?

One common problem is the efficiency of lysis, for example. Or you just do not have enough starting material.