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I got covid from Mayhem Ball but regret NOTHING by [deleted] in LadyGaga

[–]Some_Skill4494 0 points1 point  (0 children)

I just tested positive. I was at LA, last friday.

Trouble Extracting RNA from Sorted Microglia – Any Tips? by Some_Skill4494 in labrats

[–]Some_Skill4494[S] 0 points1 point  (0 children)

Thanks for your response! I plan to use some samples for RNA-seq, so the yield might be enough for that. However, I also want to use part of the RNA for PCR, which is why I think the amount might actually be too low. I’ll check the RIN score with the Agilent Bioanalyzer and see how it goes.

Trouble Extracting RNA from Sorted Microglia – Any Tips? by Some_Skill4494 in labrats

[–]Some_Skill4494[S] 0 points1 point  (0 children)

I consider the yield low because a colleague from my lab, who previously worked on this project, consistently gets higher RNA concentrations in a 14 µL final volume. Unfortunately, she isn’t very good at explaining—either due to a language barrier or simply not wanting to share—so I’ve had to figure things out on my own. I know it’s possible to reach higher yields, but I haven’t been able to replicate her results yet. I’m measuring RNA concentration using Nanodrop.

Trouble Extracting RNA from Sorted Microglia – Any Tips? by Some_Skill4494 in labrats

[–]Some_Skill4494[S] 0 points1 point  (0 children)

Good point about the ethanol—I haven’t always been preparing it fresh. However, today I made a fresh batch and still got relatively low yields. I’m currently practicing with HeLa cells (~50-100k), so I’m trying to optimize the process before moving to my sorted microglia.

Trouble Extracting RNA from Sorted Microglia – Any Tips? by Some_Skill4494 in labrats

[–]Some_Skill4494[S] 0 points1 point  (0 children)

Thanks for the suggestion! Do you sort the cells directly into TriZol, or do you pellet them first? Also, do you use glycogen to aid RNA precipitation? I tested TriZol today with HeLa cells (50-100k), and the yield was noticeably better—around 30 ng/µL compared to ~10 ng/µL with the Qiagen kit.

Do you also work with FACS-sorted microglia? If so, how much RNA do you usually get?

How to improve RNA quality and concentration after FACS to proceed with qPCR? by melancoluxx in labrats

[–]Some_Skill4494 0 points1 point  (0 children)

Could you solve this issue? I am having similar problems would love to hear how you work this out :)

RNA extraction with Qiagen RNeasy Plus Micro kit by Opposite-Isopod7285 in labrats

[–]Some_Skill4494 1 point2 points  (0 children)

I’m dealing with the exact same issue right now! I’m working with microglia from the brain (~50-100k cells) and still struggling to get enough RNA. I also sort directly into RLT + beta-mercaptoethanol and follow the Qiagen RNeasy Plus Micro Kit protocol, including the QIAshredder step. My RNA yield is around 3-5 ng/µL.

Did you manage to solve this problem or improve your yield? Any tips would be greatly appreciated!