Troubleshooting: RNA Isolation with TRIZOL Reagent
Contents
1. Troubleshooting RNA Isolation with TRIZOL Reagent Problem Low yield of RNA Possible Cause RNA not solubilized completely Sample not homogenized completely Suggested Solution To increase solubilization pipet RNA pellet repeatedly in SDS or DEPC treated water Heat sample at 55 C for 10 to 15 min Do not allow RNA pellet to dry completely Do not lyophilize or vacuum dry samples Note Clear pellet indicates over drying Do not centrifuge RNA above 12 000Xg Make sure no particulate matter remains Be sure to incubate for 5 min at room temperature after homogenization Degraded RNA Sample manipulated too much before TRIZOL Reagent addition Improper storage of RNA Frozen tissue thawed in absence of TRIZOL Reagent Process tissue immediately after removal from animal For cell culture samples minimize washing steps Add TRIZOL Reagent directly to plates Do not trypsinize cells Store isolated RNA at 70 C not 20 C Add frozen tissue to TRIZOL Reagent Low Ax 60 280 lt 1 65 Residual organic solvents in the RNA phenol chloroform Sample not homogenized with sufficient TRIZOL Reagent pH of solution is acidic Ar 60 Or Ag outside the linear range Be sure not to carry any of the organic phase with the RNA sample Precipitate the RNA again with ethanol Use 1 ml TRIZOL Reagent for up to 50 mg tissue or 106 cells Be sure to incubate sample for 5 min at room tempe2. rature after homogenization Dissolve sample in TE instead of water Dilute sample to bring absorbance into linear range RNA contains some DNA Part of the interphase was removed with the aqueous phase Insufficient TRIZOL Reagent used Cells or tissue contained organic solvents Insoluble materials were not removed before chloroform extraction Be sure not to take any of the interphase contains the DNA with the aqueous phase Use 1 ml of TRIZOL Reagent for 50 mg of tissue or 106 cells Be sure original sample does not contain organic solvents such as ethanol or DMSO Remove any particulate material before chloroform addition This material may trap DNA Problem Aqueous phase containing RNA has color Possible Cause For tissues with high fat content i e skin fat micelles do not completely separate to top of aqueous phase during centrifugation Micelles pick up red color from TRIZOL Reagent For samples with high amounts of blood some iron or hemoglobin may remain in aqueous phase giving a yellowish or maroon color Aqueous phase turns yellow upon addition of isopropanol Suggested Solution Centrifuge sample before chloroform addition and remove fat layer on top Make sure not at the upper range of sample to TRIZOL Reagent ratio Try a fresh bottle of isopropanol Note this color has not inhibited RT PCR Cells don t detach from flask after TRIZOL Reagent addition Thi
3. s can be seen with some strongly adherent cells After TRIZOL Reagent addition let cells sit 2 to 3 min Scrape cells with a rubber policeman Incu bate several minutes Collect cells and repeatedly pipet cells over flask surface Then transfer homogenate to a tube Precipitate in bottom of the tube after chloroform addi tion and centrifugation High amounts of polysaccharides or proteoglycans Remove aqueous phase and alter isopropanol precipitation step to using 0 25 volumes of 1 2 M sodium citrate 0 8 M NaCl and 0 25 volumes of isopropanol
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