Hi Susan, I also work with RNA from bovine neutrophils. I have found the best extraction method is with Trizol.

A few tips, I use ice cold propanol and I incubate in propanol overnight; I also wash with ethanol three times. Below is my protocol, I was able to get a RIN of 10 with this protocol. All the best!

RNA Extraction using TRIzol

Materials per # of samples:

30µl RNase-free water 500µl cold isopropanol

200µl chloroform 1000µl 75% ethanol

Method:

1. Add 1ml of Trizol to 5-10x106 cells in a sterile 1.5ml tube.

2. Incubate at room temperature for 5minutes

3. Add 200µl chloroform and shake for about 15seconds, incubate at room temperature for 15minutes.

4. Centrifuge @ 12,000 x g (9,000 RPM) at 4°C for 15minutes.

5. Put on ice. Transfer aqueous phase(top) to a clean tube (save the interphase containing DNA and RBCs for protein later extractions at 4°C). Be careful not to mix interphase!

6. Precipitate RNA with 500µl cold isopropanol at 4°C for 1.5hours (90mins), best to incubate overnight at -20°C

7. Keep on ice. Centrifuge for 10minutes at 4°C 12,000 x g (9,000 RPM), dispose supernatant, RNA is a gel-like pellet at the bottom.

8. Wash pellet with 1000µl of 75% ethanol (do this 3x), keep on ice.

9. Vortex and centrifuge for 5minutes at 4°C 7,500 x g (8, 000 RPM), discard supernatant by dapping with clean wipes (important!)

10. Air dry pellet for about 5-10 minutes

11. Dissolve RNA in 10-30µl RNase-free water, mix with pipette. Put on ice.

12. Keep on ice. Check for concentration using a Agilent Bioanalyzer or Nanodrop

Try using a Qiagen cell shredder as indicated in the RNAeasy protocol this tends to increase the yield !

• Ebo has provided an excellent answer
• I would only add to it by saying that when you precipitate from rate limiting amounts of material and therefore anticipate < 1ug of total RNA, add 1ul of 1mg/ml to 20mg/ml molecular grade glycogen as a carrier
• Any where in this concentration range is permissive: Provding the final concentration of glycogen in your alcohol based solution is > 1ug/ml your pellet will be much more conspicuous with a carrier (opaque and larger until dryed) and therefore easier to wash with 70% ethanol. Recovery of RNA precipitated with carrier like glycogen is invariably > 90% as opposed to (typically ) 70% to 90% without carrier which makes all the difference for small amounts of starting material
• I also think as Ebo has suggeted that overnight precipitation will always improve yield but you also incrase the amount of salt you bring down, especially with isopropanol (as opposed to 3 volumes of ethanol as an alternative)
• Nevertheless, providing you perform a vigorous washing procedure with 70% ethanol as described by Ebo you will remove this salt

Hi, thanks for your response. I tried the isopropanol precipitation, but still I didn't get any RNA. I was very careful and started with new solutions to be sure I did not have RNase contamination.

I'll try with the glycogen and/or increasing the number of cells.

Eboghoye Eluka , do you always work with 5 x10^6 cells?

Dear Susan

• What is your final elution volume as well as concentration ?
• Are you still using Trizol and RNeasy still or one or other as I suggested ?
• Could you give me a summary of how your harvest RNA from neutrophils ?
• What is your total yield of RNA ?
• 5 X 10(6) is not uncommon for neutrophils derived from leucocytes but that number of cells is definitely very small

you can use RNA product in state of sample an run the same protocol