I am using rabbit cornea to do LMD and i am collecting the laser dissected fragments directly in the RNA lysis buffer as suggested in the RNeasy micro kit from qiagen where they have a protocol for LMD RNA extraction. However the final RNA yield is very low in range of 30-40ng. Is it better to collect the fragments in RNA stabilization buffer, if so how to proceed for downstream processing? Also when i collect my fragments in RNA lysis buffer, i let them at RT. Kindly share any experiences regarding this.
Hi
On of the main issues is how you store your samples prior to LCD. I use to preserve all my samples in -80 and I process them in paraffin 24 hours prior to the use of LCM. You could also increase the number of sections per sample during the extraction to yield higher amount. It would be better if you placed your membranes in ice during dissection if your are processing many samples.
I hope this is helpful.
Regards,
Bassem
Hi
On of the main issues is how you store your samples prior to LCD. I use to preserve all my samples in -80 and I process them in paraffin 24 hours prior to the use of LCM. You could also increase the number of sections per sample during the extraction to yield higher amount. It would be better if you placed your membranes in ice during dissection if your are processing many samples.
I hope this is helpful.
Regards,
Bassem
30-40 ng of RNA = about 1750 cells' worth of RNA, which for LMD is fairly good actually. Predicated on the idea that a mammalian cell contains about 20 pg of total RNA. You may already be OK with your yield, depending on how much you dilute such samples prior to RT-qPCR analysis. Keep your samples cold when at all possible. My gut feeling is that you should go ahead and try one-step qPCR with the RNA you have (after DNAse treatment of course). A little trial and error goes a long way here. See the attached publication for other details.
Hi Bassem, Thanks for the reply. My tissue is immediately flash frozen and always store in dry ice, when i use it for sectioning. I have increased the number of sections each time to increase the RNA yield. Given the limitation of the technique, i am trying my best to have as high RNA yield as possible.
Hi Jack, Thanks for the description. As you said i may getting the better yield. Eventhough my RNA amount is low, i have used them for cDNA prep and downstream qPCR reaction and they all went well. When i collect the LCM fragments directly in the RNA lysis buffer, i let them stand in RT, not sure i can store them at 4 degree. I will read the article you linked. Hope i will get some hints to improve the yield.
The lysis buffer should also inactivate/denature any RNAses as well correct? So, your RNA is likely safe at room temperature for longer than you might think (you can always add a little RNAseOUT if unsure). Your yield is probably maximal already - only slight mechanical agitation (vortexing) with elevated temperature (50C) might help - but, you may already be getting all the RNA you can as is.
Yes Jack, I was also thinking the same and hence allowed it in RT. Thanks for the tips.
I have used the ROCHE High Pure RNA Isolation Kit sucessfully for isolation of RNA from small amounts of cells.
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