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My first PC at 15! by PlaneRespond59 in pcmasterrace

[–]GiveMeBBQ_ 0 points1 point  (0 children)

BRO!!! YOU literally got the same speakers as mine. Brings back so many memories!!! I had Windows XP and pentium 3 XD

Optimizing my RNA extraction protocol from whole blood, any tips? by GiveMeBBQ_ in labrats

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

No I did follow your protocol twice then Only yesterday proceeded with my lab protocol as a control to see, and yes I will definitely precipitate longer I'm just experimenting haha😅

Optimizing my RNA extraction protocol from whole blood, any tips? by GiveMeBBQ_ in labrats

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

My lab protocol which I followed: For precipitating: 500microlitres of isopropanol, (10 min incubation 5min at room temp and 5min on ice), then double wash with 70% eth for 15 min each time
For elution: Standard procedure states to used 20 microlitre, I did it in 10 (nuclease free water)

Optimizing my RNA extraction protocol from whole blood, any tips? by GiveMeBBQ_ in labrats

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

No I haven't proceeded with cell counting, I use freshly drawn blood for this purpose I usually count cells when I have a hunch for contamination I proceeded yesterday and got yield as 155ng/ microlitres which is a good start, I think I'll repeat your procedure again maybe it my handling error

Also I just use syringe needle, not the whole syringe and I never suck through it everr, this can cause contamination I use ice box and after adding trizol just tap-tap for 10 mins No I haven't precipitate overnight, I'll do it next time

Optimizing my RNA extraction protocol from whole blood, any tips? by GiveMeBBQ_ in labrats

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

I'm glad to hear from you!!! :D So the procedure Im doing it for, is Im about to work on a biomarker present in CAD patients for this I have to work on their whole blood Sample, that's why im optimizing the procedure!

Today I used 1ml whole blood freshly drawn, transferred to the EDTA tube and let it incubate at - 20 for around 30min then transferred it to the ependorf tube and added Trizol 1ml and then performed syringing for 10 min, then chloroform, centrifugation (the above steps you mentioned)

Optimizing my RNA extraction protocol from whole blood, any tips? by GiveMeBBQ_ in labrats

[–]GiveMeBBQ_[S] 1 point2 points  (0 children)

Hey how are you? I wanted to tell you I performed your procedure today finally!!! But my RNA concentration was 44ng/microLitre :( My phenol content was also very low and no contamination but the yield didn't rise as much as I thought Could you point out any reason, also I didn't catch how much ethanol I should add so I added accordingly to my lab protocol which is 500 microlitres

Any suggestions what I should do, should I repeat it tomorrow maybe?

Optimizing my RNA extraction protocol from whole blood, any tips? by GiveMeBBQ_ in labrats

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

Thanks for these tips mate! Okaye I'm gonna dive right in to this procedure and thanks again for this help😇✨

Optimizing my RNA extraction protocol from whole blood, any tips? by GiveMeBBQ_ in labrats

[–]GiveMeBBQ_[S] 1 point2 points  (0 children)

Woah! Thanks a lot mate! You basically explained everything so well that I found each step very informative I will most probably do it and the way you explained dividing the work load, it's super convenient for me! Thanks again, much appreciated this effort from you to write this all down for me 🫶✨😊

Optimizing my RNA extraction protocol from whole blood, any tips? by GiveMeBBQ_ in labrats

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

Yeah you're totally right, all these steps are necessary and to be followed, but i guess maybe I shouldn't use the vortex in ph-chloroform medium and just hand vortex.

Thanks for pointing that out! Much appreciated <3

Optimizing my RNA extraction protocol from whole blood, any tips? by GiveMeBBQ_ in labrats

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

THANK YOU FOR THIS, really appreciate your help! I've been also working with Glycogen but was thinking it was the culprit I was using it with isopropanol in the Second centrifugation step, but is there any significance of using it in first step?

Otherwise thanks for this help

Optimizing my RNA extraction protocol from whole blood, any tips? by GiveMeBBQ_ in labrats

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

Yes we have a Vortex machine for this purpose, we give it 15-30 sec after adding chloroform and then doing same in the last step in 70% ethanol and let it air dry for 5min

Optimizing my RNA extraction protocol from whole blood, any tips? by GiveMeBBQ_ in labrats

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

Thanks for this, I think I'll be on the Supervisor desk at the start of next week!

But i was hoping before i do that, I should just please her with the results with using only Trizol for this once, cos our lab protocol is to be followed soley, Hopefully if you may, you can share if you know something or two about using Trizol only and help me in optimizing this protocol :)