mRNA pico - Can't determine exact RNA size

[SeqVicious]

Hi all,

I'm performing an altered RNAseq library preparation where I need to start from between 80 and 110 bp of fragmented mRNA. I ran the samples on a Urea-PAGE gel and extracted my sizes. I ran them on a bioanalyzer and I'm having trouble determining the average size of my peak. It's under 200bp and greater than 25. I'm worried that they're all centered around 150 or so. Does anyone know how to tell where the size is centered using these two markers?

[nucacidhunter]

You need to run dilution of your RNA as the Chip seems overloaded. To get average size you need to select a range flanking your fragments.

[SeqVicious]

Why does this seem overloaded? It's only 20ng/ul. How do I select a range? Can I do this in the actual data on the computer software?

[SeqVicious]

Also, I should note that I have the sequencing center at my university do the bioanalyzer. I can try asking them if I can get the data in a different format.

[nucacidhunter]

I would suggest looking at Pico Chip manual for its specification. Overloading is indicated by the lower height of marker in comparison to sample. You can specify a range in software by opening the file/clicking on sample name which will open electropherogram/clicking on Region Table at the bottom of screen/ right clicking on plot area and selecting Add Region. The region bars can be moved to flank the sample area.

[kerplunk412]

I believe the maximum concentration for a RNA Pico chip is 10 ng/uL. They are very sensitive. That being said, Bioanalyzer quantification isn't all that accurate anyway, and it seems size is what you are really interested in.

Contact your sequencing facility and nicely ask them if they can get you a report that also indicates the size of you sample. They can do it using the steps that nucacidhunter suggested in the Bioanalyzer software.

Also, at the concentration you have a TBE-Urea PAGE gel stained with SYBR Gold or maybe even EtBr should give you a pretty good indication of the size. It is just as reliable as the Bioanalyzer, maybe even more so.