Hello,
I am having issues isolating quality RNA for gene expression profiling.
I isolate total RNA from samples stored in Trizol followed by an extra purification step via Qiagen RNeasy Minielute columns (Cat. # 74204). I obtain small amounts (20-240ng) of RNA according to a nanodrop with a RIN number greater than 9 on the bioanalyzer.
However, on the nanodrop I get a peak a 230 (charcterisitc "shoulder" at 225nm), and a bump and 270nm. I have heard that these two peak problems are normal when isolating small amounts of RNA via Trizol and near impossible to remove.
I would like to use the RNA to make libraries for gene expression profiling on the Illumina HiSEQ but do not want to submit low quality RNA. Is there a way that I can check ahead of time to make sure that whatever the source of these peaks will not affect library construction (RT-PCR?).
I would greatly appreciate any ideas on how to further purify my RNA (butanol/ether experiences?), any past experiences as to whether or not such nanodrop readings predicted poor library construction, and other ways to better purify small samples already stored in Trizol.
Thank you for your time,
Katie
I am having issues isolating quality RNA for gene expression profiling.
I isolate total RNA from samples stored in Trizol followed by an extra purification step via Qiagen RNeasy Minielute columns (Cat. # 74204). I obtain small amounts (20-240ng) of RNA according to a nanodrop with a RIN number greater than 9 on the bioanalyzer.
However, on the nanodrop I get a peak a 230 (charcterisitc "shoulder" at 225nm), and a bump and 270nm. I have heard that these two peak problems are normal when isolating small amounts of RNA via Trizol and near impossible to remove.
I would like to use the RNA to make libraries for gene expression profiling on the Illumina HiSEQ but do not want to submit low quality RNA. Is there a way that I can check ahead of time to make sure that whatever the source of these peaks will not affect library construction (RT-PCR?).
I would greatly appreciate any ideas on how to further purify my RNA (butanol/ether experiences?), any past experiences as to whether or not such nanodrop readings predicted poor library construction, and other ways to better purify small samples already stored in Trizol.
Thank you for your time,
Katie
Comment