Hi, I have been struggling with a weird issue for my RNA samples. I used the Qiagen RNA plus mini kit. For some reason no matter how careful I am with the washes, when I measure the RNA, I get really low values for the 260/230 ratio, on average around 0.5 or below. I follow all the steps suggested in the kit.. I thought that it must be the Ethanol which causes such ratios so I try to adequately dry the column with an extra spin.. but that does not seem to help.
I checked the quality of my RNA on the Bioanalyzer and it seems the quality is fine. Even the yield of RNA from my samples is decent .. ( I grow cells in 6 well plates ).
Carryover of chaotropic guanidium salts is pretty common with kits, in my experience.
If your yield is good, you can usually clean up your RNA by precipitation:
Make up vol to 500ul with nuclease free water
Add 0.1 vols (50ul) of 3M sodium acetate, pH 5.5
Add 10ug of glycogen (optional: increases recovery yield)
Mix.
Add 1 vol (500ul) of room temperature isopropanol.
Incubate at RT for 20mins, spin at max in a chilled benchtop (4 deg) for 15 mins.
Wash 2x with ice cold 75% ethanol (add ~300ul gently to wash over the pellet, spin after each addition).
Air dry for 15mins or so
Redissolve in water.
This approach will generally lose ~20-25% of your RNA, but 90-95% of your salt, giving you much cleaner, usable RNA.
Room temp is important, btw: cold isopropanol will precipitate salt too, which you really don't want.
A low 260/230 is often an indication of chaotrophic binding salts eluting with the rna. Try using fewer cell and making sure that lysis is complete ( this stops blocking the column and retention of salts) and wash well with warm wash buffer.Dissolve the rna in TE not water as pH can have a very large effect on measuring the amount of rna on nanodrop
Carryover of chaotropic guanidium salts is pretty common with kits, in my experience.
If your yield is good, you can usually clean up your RNA by precipitation:
Make up vol to 500ul with nuclease free water
Add 0.1 vols (50ul) of 3M sodium acetate, pH 5.5
Add 10ug of glycogen (optional: increases recovery yield)
Mix.
Add 1 vol (500ul) of room temperature isopropanol.
Incubate at RT for 20mins, spin at max in a chilled benchtop (4 deg) for 15 mins.
Wash 2x with ice cold 75% ethanol (add ~300ul gently to wash over the pellet, spin after each addition).
Air dry for 15mins or so
Redissolve in water.
This approach will generally lose ~20-25% of your RNA, but 90-95% of your salt, giving you much cleaner, usable RNA.
Room temp is important, btw: cold isopropanol will precipitate salt too, which you really don't want.
I would recommend doing the isolation of RNA manually. Its more easy with the cell lines than in the tissue samples. You can also dissolve your RNA in 0.1% DEPC treated water.
Carbohydrates and phenol have absorbance around 230 nm. If you have a low 260/230 ratio, the cause is most probably phenol contamination. Make sure you evaporate all the ethanol. I agree with John Hildyard you can wash your RNA sample to get rid of any phenol residual.
Ratio of 260/230 shows chaotropic salts, reduce on cells, precipitate your RNA and dissolve in TE.
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