Hi all,
I am new to the forum, so I will make this my introduction and first question to the forum. I am currently working with the TU-tagging method (TU-tagging: cell type specific RNA isolation from intact complex tissues, Miller et. al. 2009) to isolate RNA from cells in the Drosophila CNS. The protocol has been working when assessed by real time PCR, however there are about 3 times during the isolation of the "tagged" RNA that one has to clean up the RNA. The workflow looks like the following:
1) Isolate total RNA via Trizol and column clean-up or linear acrylamide precipitation -> 2) Isolate mRNA with Dynabeads -> 3) Label Thiol tagged RNA via Biotin-Thiol coupling -> 4) Precipitate RNA -> 5) Purify biotin labelled RNA with magnetic streptavadin beads -> 6) Precipitate RNA again -> 7) Proceed to cDNA amplification step (NuGen) and ultimately on to Seq. library preparation
I cannot afford to loose RNA at each of the clean-up steps so I have decided to move away from minElute columns. In my hands, I find the columns are a little inconsistent and sometimes I will lose up to 50% of my RNA. Thus, I have settled on linear acrylamide, as it supposedly can recover almost 100% of the input RNA and is compatible with all following downstream steps.
I have a few questions regarding the use of linear acyrlamide assisted precipitation
1) In this protocol I have to precipitate the same RNA sample about 3 times in succession. Do I have to add linear acrylamide at all the precipitation steps, or just the initial step? Each step will be precipitating the RNA from roughly the same total volume.
2) Does anyone have any hints for how to not loose/visualize the pellet of RNA? I can rarely see my pellet and on the last step where I isolate the biotin labelled RNA, I may only have 5 ng of RNA (maybe less), so the pellet is going to be really hard to see.
Thanks
Sean Speese
Research Assistant Professor
OHSU - Jungers Center for Neurosciences Research
I am new to the forum, so I will make this my introduction and first question to the forum. I am currently working with the TU-tagging method (TU-tagging: cell type specific RNA isolation from intact complex tissues, Miller et. al. 2009) to isolate RNA from cells in the Drosophila CNS. The protocol has been working when assessed by real time PCR, however there are about 3 times during the isolation of the "tagged" RNA that one has to clean up the RNA. The workflow looks like the following:
1) Isolate total RNA via Trizol and column clean-up or linear acrylamide precipitation -> 2) Isolate mRNA with Dynabeads -> 3) Label Thiol tagged RNA via Biotin-Thiol coupling -> 4) Precipitate RNA -> 5) Purify biotin labelled RNA with magnetic streptavadin beads -> 6) Precipitate RNA again -> 7) Proceed to cDNA amplification step (NuGen) and ultimately on to Seq. library preparation
I cannot afford to loose RNA at each of the clean-up steps so I have decided to move away from minElute columns. In my hands, I find the columns are a little inconsistent and sometimes I will lose up to 50% of my RNA. Thus, I have settled on linear acrylamide, as it supposedly can recover almost 100% of the input RNA and is compatible with all following downstream steps.
I have a few questions regarding the use of linear acyrlamide assisted precipitation
1) In this protocol I have to precipitate the same RNA sample about 3 times in succession. Do I have to add linear acrylamide at all the precipitation steps, or just the initial step? Each step will be precipitating the RNA from roughly the same total volume.
2) Does anyone have any hints for how to not loose/visualize the pellet of RNA? I can rarely see my pellet and on the last step where I isolate the biotin labelled RNA, I may only have 5 ng of RNA (maybe less), so the pellet is going to be really hard to see.
Thanks
Sean Speese
Research Assistant Professor
OHSU - Jungers Center for Neurosciences Research
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