1) Poor Yield Of Total RNA Is Mostly Due To Incomplete Sample Lysis, Thus Leading To Incomplete Release Of RNA. Since Good Yield And Good Quality Of Total RNA Are Only Assured When Sample Is Properly Handled And Lysed Completely, Do Not Use More Than The Amount Of Sample Suggested In The Protocol.
2) Thorough Celluar Disruption Is Critical For High RNA Quality And Yield. RNA That Is Trapped In Intact Cells Is Often Removed With Cellular Debris And Is Unavailable For Subsequent Isolation. Therefore It Is Crucial To Choose The Disruption Method Best Suited To A Specific Tissue Or Organism To Maximize Yield. Mechnical Cell Disruption Techniques Include Grinding, Homogenization WithDouce Or Rotor-Stator Homogenizers (Polytron), Vortexing, Sonciation, And Use Of Bead And Freezer. Complete Disruption Of Some Tissues May Require Using A Combination Of These Techniques. Rotor-Stator Homogenizers, Alone Or In Conjunction With Other Disruption Techniques, Generally Result In Higher RNA Yields Than Other Types Of Homogenizer.
3) Another Most Frequent Cause Of Low RNA Yield Is Overloading The Column, Which Can Cause The Column To Clog Or Can Prevent The RNA From Binding To The Membrane Efficiently. Methods That Reduce Viscosity, Such As Reducing Sample Amount, Diluting The Viscous Lysate With RX Buffer, Disrupting The Sample More Extensively, And Centrifuging To Remove Insoluble Remains, Will Increase RNA Yield. If Yields Are Still Lower Than Expected, Consider Diluting The Clarified Lysate And Splitting Loading Into Two Columns, Which Will Further Reduce The Concentration Of Contaminants And Improve RNA Binding And Recovery.
4) When RNA Is To Be Eluted, Make Sure That RNase-Free DdH2O Is Added Onto The Membrane And Penetrate Into It. If DdH2O Still Retains On The Membrane, Pulse Centrifuge The Column For A Few Seconds To Drag It Into The Membrane.
5) Eluting The Column Twice Can Result In A Higher RNA Recovery, Especially When Expected RNA Yield Is More Than 30 Mg.
