Isolate at least 10 million murine B cells, add 1 mL of TRIzol to the pellet, proceed with usual TRIzol procedure, and resolubilize the final pellet in 50 uL nuclease-free water.

Note:  at the point in the TRIzol RNA isolation procedure where you save the top (RNA-containing) aqueous layer - be sure to grab about exactly 550 uL of it. The layer will actually be about 630 uL, but only grab 550 uL -- to avoid the white interface layer (gDNA and protein) entirely.  DNAse-treat the RNA with TURBO DNA-free DNAse, and there you have it.   If you measure the concentration of the RNA on a spec, measure it at a 1:25 dilution, and be sure to zero the spec or NanoDrop with a sample of water that has been carried through the entire isolation procedures in parallel with the mouse B cell RNA isolation samples. Blanking the spec correctly (post DNase treatment), is key.

Thanks JACK 

I wanna to explain more about my procedure , first I isolated the RNA from B cell pellet  which were treated with RNA later ICE overnight , the B cells around 20 million , the TRizol  procedure is used with all precaution to prevent DNA  contamination , I got 80 ng/ul RNA and 600ng/ul DNA but once I checked the RNA integrity ın 1.1 % agarose  in TAE  I could not visualize the RNA bands although I visualize the clear and bright RNA bands form other human cell line even the RNA con from 50ng - 100 ng / ul 

Did you isolate the RNA from B cells ?

what is the possible problem in my  procedure 

Thanks in advance

A lot of detail missing in your original question.  But, RNA later ICE is for adding to samples that have already been frozen and are being thawed out -- would have been better to place the pellets first in 'RNA later' then freeze.   But, best of all is to put the pellets directly into 1 mL of TRIzol before freezing.  The RNA later ICE would affect your gel runs -- and for RNA you would need to do a 1% agarose/formaldehyde-based gel format to visualize bands correctly.  Also - you speak of DNA as well (isolated by TRIzol) -- so I am not sure what TRIzol protocol you are following.

If you did not homogenize your B cell pellets directly in TRIzol before freezing you could have released RNAses during the storage/freeze-thaw process.  Also - if you have access to a Bioanalyzer 2100 - that would be better to check RNA integrity.  Were the human RNA samples you speak of isolated the exact same way as your murine B cells?  Placing pellet of B cells into TRIzol is your best bet prior to RNA isolation here...

I have performed numerous buffy coat RNA isolations which is a direct correlate to what you are attempting to do here (albeit with mouse B cells) - with great success as described in my post above.  Good Luck!

 The RNA from human cell line like MKN cells is used  as control to check RNA integrity . yes I used the RNA later ice for frozen pellets   . However I used the Ribo pure kit based on TRIzol  reagent and chloroform , then DNA precipitation  from  interphase  by using 100 % ethanol and DNA wash by (0.1 M sodium citrate in 10% ethanol, pH 8.5).

Thanks

Hello Sawsan,

Protocols for RNA isolation from cells are well established. All one has to do is to follow them meticulously. Just do it one more time; I am sure you ll get it to work. You compared the RNA yield from another cell type. Did you do purification of RNA from two cell types in parallel?

20 million mouse B cells (CD19+) should contain at least 25 ug total RNA (16-40 ug), so, you are most likely dealing with a much smaller amount of total RNA (in B cell RNA isolations) when compared with the total RNA yield you are getting from the human MKN cell line you are also working with. You may be dealing with up to 100X less RNA from B cells; especially if also 'unstimulated.'

Another possibly helpful RG thread on this is at:

https://www.researchgate.net/post/RNA_extraction_of_human_T_cells2

Both RNA Later and Trizol extractions have worked for me, with far fewer B cells  (as few as 5,000) as long as the target is amplified by nested PCR / at least two amplification runs.  

Dear Jean

Even the RNA is low you can use it for PCR but for RNA seq or Microarray  we need pure and concentrated RNA  and in my case I could not isolate  concentrated RNA and I could not visualize it on gel  even 80ng/ul ,

Oh, right - I should have realized.  

You may want to check the viability of your sorted B cells. Mouse B cells are not the hardiest cell type and they tend to die quite easily. MACS is quite effective but if isolating by FACS, you need to use a larger diameter nozzle which slows the flow rate and decreases the cell stress. If your cells are in the early stage of apoptosis, the RNA may be of low quality and your isolation procedure may not be the problem. I would check the viability of the B cells at the time just prior to when you would begin RNA isolation after the B cell or B cell subset of interest has been purified.

Dear Daniel

The B cells were isolated by MACS and I got around 80 % purity , additionally I checked the B cell viability By trypan blue or  7ADD, the viability is around 85% ,

Do you have any further suggestion ?

You might look at cleaved PARP and activated caspase 3 to investigate whether cells are in early apoptosis. Although the cells appeared viable in your assessment, perhaps they were starting down the road to cell death which could hurt your RNA quality. Otherwise, your approach seems reasonable and you may just try to repeat it some more times.

Dear Daniel  

Thanks in advance