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RNA extraction from human monocyte..why low yield? - (May/27/2008 )

Helo, can anyone give n extra info about rna extraction especially from human monocytes..can u see two clear bands of 18s and 28s if u isolate them from 1000 to 100000 monocyte cells.

usually cultured cells didnt proliferate well in my lab (dunno why) so the cells is quite limited to isolate the rna..as i would proceed the rna to do reverse transcriptase pcr, the yield is quite disappointing as it is very difficult to get a sharp clear band (the pcr product)

so, i wonder it is because of the quality rna factor or some other else (i dont think the problem lies on rt pcr as i am convinced that the reaction is already optimized)

is there any suggestion on that... thanx in advance to whoever person that respond to my make-me-ponder matter. happy.gif

-vertical-

QUOTE (vertical @ May 27 2008, 11:11 AM)
Helo, can anyone give n extra info about rna extraction especially from human monocytes..can u see two clear bands of 18s and 28s if u isolate them from 1000 to 100000 monocyte cells.

usually cultured cells didnt proliferate well in my lab (dunno why) so the cells is quite limited to isolate the rna..as i would proceed the rna to do reverse transcriptase pcr, the yield is quite disappointing as it is very difficult to get a sharp clear band (the pcr product)

so, i wonder it is because of the quality rna factor or some other else (i dont think the problem lies on rt pcr as i am convinced that the reaction is already optimized)

is there any suggestion on that... thanx in advance to whoever person that respond to my make-me-ponder matter. happy.gif


Hi vertical
I deal with a similar problem. I extract RNA from primary cells - monocytes separated from blood by magnetic beads. I tried RNeasy Kit and QiaAmp RNA Blood Mini Kit, both from Qiagen and both with the same dissapointing results. I rarely can see two clear 18S and 28S bands and the yields are totaly unsatisfactory. :-( Unfortunately, I have no advice for you. It seems that monocytes are sort of freaky cells when it comes to RNA extraction.
Paja

-Paja-

Hi, i am using Promega kit to extract mRNA... But how do i know if cell lysis had occur? Does vortexing it help to lyse the cell?

-Jash-San-

QUOTE (Jash-San @ Jun 17 2008, 08:43 AM)
Hi, i am using Promega kit to extract mRNA... But how do i know if cell lysis had occur? Does vortexing it help to lyse the cell?



my lab do have the kit..but still the result is unsatisfactory...we cant see that the cells are already lysed, but from my experience extracting dna from cells, if you put lysis buffer then you can incubate it overnight to fully lyse the cell...

but rna cant do overnight...5 min is enough to incubate cells in lysis buffer (RLA)...and yes you can vortex the cell to help lysing cell (i get this tips from my colleuges..but you can ignore this if you want as i myself cant get any good result though.. wink.gif

-vertical-

Paja you also did monocyte culture? you are a msc of phd student? is your culture ok? im also having contamination problem...(~sigh..i got so many problem sad.gif ) Can you share some of your virtues in dealing with monocyte cullture...

my email add is afeefah_yusof@yahoo.com

-vertical-

can you try to grow more flask and pool them?

I extract RNA from epithelial cells using the protocol below:
http://www.path.cam.ac.uk/~toxo/Protocols/...20ISOLATION.pdf

the protocol always give me clean RNA and good RIN no of RNA.
I'm not sure if this protocol could help, but i just love to share this useful protocol to all my friends! tongue.gif

-sanjiun81-

Hi guys, please try AquaRNA. It'll work wonder for you and you could ask for a free trial sample.

-chessplayer-