This System Is Not Suitable For Samples Of Liquid Form Such As Whole Blood. However, It Can Extract RNA From Buffy Coat. This System Cannot Be Used For Liquid Samples Because Liquid In Sample Will Dilute Out RX Buffer, Thus Reducing Its Lysis And RNase Inactivating Ability. Since Only White Blood Cells Contain RNA, One Should Only Use Buffy Coat For RNA Extraction. Buffy Coat For RNA Extraction Should Be Prepared From Fresh Blood Sample.

Three Critical Steps, If Not Done Well Can Cause RNA Degradation. They Are 1) Handling And Storing Of Samples, 2) Disruption Of Samples, 3) Storage Of Eluted RNA. 1) Most Animal Tissues Can Be Processed Fresh (Unfrozen). It Is Important To Keep Fresh Tissue Cold And To Process It Quickly (Within 30 Minutes) After Dissecting. If Samples Cannot Be Processed Immediately, It Should Be Flash Frozen In Liquid Nitrogen And Stored At -80°C. Samples Should Be Handled With RNase-Free Tools. 2) When Sample Is Disrupted, Disruption Needs To Be Fast And Thorough. Slow Disruption, E.G. Placing Cells Or Tissue In RX Buffer Without Any Additional Physical Shearing, May Result In RNA Degradation By Endogenous RNase Released Internally, Yet Still Inaccessible To The Protein Denaturant In RX Buffer. 3) After Elution Of RNA With RNase-Free DdH2O Provided In The System, Store RNA At -80°C. Degradation Of RNA May Also Occur During Loading Into A Gel, Use Gel And Fresh Running Buffer Prepared Using DEPC-Treated DdH2O As Well As Properly Cleaned Gel Tray And Tank For Electrophoresis. Adding EtBr Directly Into The Gel Can Also Avoid Possible Degradation Of RNA That May Occur During Gel Staining.

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.

No, Some Genomic DNA (And Plasmid DNA, If Present) Can Be Co-Purified With RNA. DNA Can Be Removed By Adding RNase-Free DNase I To The RNA Sample. DNase I Can Then Be Removed By Phenol/Chloroform Extraction.

It Is Not Impossible To Extract RNA From It, But It Involves Quite A Lot Difficulty And The RNA Yield Is Very Low That It Is Only Suitable For RT-PCR. The Preserved Conditions Of The Paraffin-Embedded Tissue Are Most Critical. First, If The Tissue Had Been Fixed For More Than 24 Hours, RNA Experienced Modification That Cannot Be Used For RT-PCR Anymore. Second, The Older The Tissue Block Is, The Poorer The RNA Yield Is. Tissue Blocks Stored For More Than 6 Months Are Thus Not Suitable. Third, Only Short-Template RT-PCR (< 400-Bp) Can Be Performed. Fourth, Since Only A Tiny Bit Of Tissue Sample Is Processed, It Is Of Course Hard To Disrupt The Sample Mechanically. In This Case, Proteinase K Should Be Used To Digest The Sample. However, Proteinase K Is Not Provided In The Kit. Fifth, Since Total RNA Extracted Is So Little, RNA Carrier (Like The One Used In Viral RNA Extraction System) Should Be Added To Enhance RNA Recovery. Sixth, Paraffin Needs To Be Removed Before The Tissue Is To Be Disrupted. Immunoport Total RNA Extraction System Is Not Tuned For Processing Paraffin-Embedded Tissues.