rna extraction from chlorella vulgaris

[najib abdellaoui]

hello everyone 

i am working on chlorella vulgaris and trying to extract RNA from it using different methods:

• i tried to use liquid nitrogen combined with trizol,
• i tried to use plant kit from invitrogen,

however, i couldn't get good results when i run the RNA sample on gel. i couldn't get the 28 and 18S bands. i got just a smear at the bottom of the gel.

can someone help me for the rna extraction

thank you.

[John Ladasky]

Have you worked with RNA before? Do you have a sample which you have used before to extract RNA as a positive control?

 Is your work station clean?  Do you wear gloves?  Do you use filter tips on your pipets?  RNAse is everywhere.  A smear at the bottom the gel suggests degraded RNA.

[najib abdellaoui]

before using the work station, i clean it, i wear gloves and worked with RNA before using animal cells and fish organs. for the positive control, in the lab they didn't  extract RNA before, so there is no positive control for RNA. for the filter tips, we don't have them recuse of budget limitation. 

[Reuben Brown]

You need to work really cold. I spin down 50ml of culture then LN2 dip and freeze dry. I keep the sample cold as soon as I take it out of the freeze drier. 

Put in 1-3 4-5mm stainless bearings and shake at 30hz for 1min in a frozen block. 

Then straight into LN2 and put in warm lysis buffer (SDS or CTAB based) 

I find that working as cold as possible cleaned up my microalgae rna heaps! 

If you dont have freeze dryer and tissuelyser then you could try frezze thaw in cold lysis buffer with heaps of prot K