I just did my first RNA extraction from tissue using a trizol-like reagent called "Tranzol". I must have eluted it to 70ul final volume but I'm having poor yields of approximately 150ng/ul, low A260/280 ratios (peak is at 270nm and there's something carrying over giving high absorbance below 230nm).
I was using two eyes from a mouse. After removing muscles and optic nerve I sunked them in liquid nitrogen and proceded to smash them up on a mortar, I collected the pink slime with a spatula and added 1ml of Tranzol. After that I carried over with the usual protocol separating with chloroform, precipitating with isopropanol and washing with 75% EtOH (precipitate was barely visible at best). I was unable to completely dry the tubes, there was about 20ul of watery EtOH left that wouldn't evaporate.
Suggestions?
EDIT: apparently chloroform was contaminated, after switching chloroform it worked well.
You can use glycogen as RNA carrier to improve RNA precipitation.
However, I agree with Kamil that 150 ng/ul is sufficient as most polymerases need around 1 ug for reverse transcription.
How long did you allow the RNA to precipitate? Isopropanol precipitations are more difficult than ethanol as they produce a more translucent pellet. We have success precipitating overnight at -20*C. In addition, as Miriam mentioned, glycogen is great for improve RNA precipitation (they even make glycogen with a blue dye, glycoblue, that helps identify your pellet).
20ul ethanol left over is quite a bit. If it's not drying at room temp, you can try drying at 37*C, with a watchful eye: check your pellet every 3-5 mins to prevent over drying. You can also manually remove excess ethanol using a pipet. Both excess ethanol and leftover phenol will cause aberrant peaks at 230 and 270 nm.
I have never used this reagent, but I can say that when I have gotten a low RNA yield, it's usually because the cells are not thoroughly lysed. Following up on Samatha's point, you can remove ethanol with a pipette if you are careful not to remove the pellet. I have dried it in a vacuum oven, but I don't recommend this, as you can easily overdry it. If you can post an image of the peaks you're seeing on the nanodrop, it would provide more information as to what kind of contamination you might have. I have attached the nanodrop booklet with images depicting what contamination peaks with reagents look like.
http://www.nanodrop.com/Library/T042-NanoDrop-Spectrophotometers-Nucleic-Acid-Purity-Ratios.pdf
I think maybe reduce your elution buffer (presume is DNAse free water?) as 70 ul its quite a lot to concentrate your RNA. Try eluting in about 50 ul if possible.. also what do you plan to do with your RNA? RT-qPCR or RNA-seq? as 150 ng/ul would be sufficient enough for RT-qPCR, need about 0.5 ug -1.0 ug
Secondly the 20 ul of watery EtOH left that wouldn't evaporate might also be another technical problem, maybe try tap through your tubes consecutively before air dry them on a paper towel. In addition, do you also do DNAse treat your extracted RNA after? as the low ratios you getting might be due to DNA contamination.
I really can't find any problems in the protocol you followed. The concentration you are getting is not low. I usually dissolve the pellets in 20 uL DEPC water and if the ethanol is not drying completely you can carefully absorb using a fine sterile tissue paper in the eppendorf by putting inside manually. Hope you try this
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