1. RNA extraction

2. Nucleic Acid Chemistry DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) store and transfer genetic information in living organisms. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) store and transfer genetic information in living organisms. DNA: DNA: – major constituent of the nucleus – stable representation of an organism’s complete genetic makeup RNA: RNA: – found in the nucleus and the cytoplasm – key to information flow within a cell

3. Structure of RNA nucleotides

4. The Central Dogma

5. Video 1 The central dogma YouTube - From RNA to Protein Synthesis_2 workshop.flv YouTube - From RNA to Protein Synthesis_2 workshop.flv

6. Cellular “ total ” RNA Messenger RNA (mRNA): 1-5% Messenger RNA (mRNA): 1-5% Serves as a template for protein synthesis Ribosomal RNA (rRNA): >80% Ribosomal RNA (rRNA): >80% Structural component of ribosomes Transfer RNA (tRNA): 10-15% Transfer RNA (tRNA): 10-15% Translates mRNA information into the appropriate amino acid  Small RNAs

7. Total RNA Purification Goal: Isolate RNA from other cellular components Goal: Isolate RNA from other cellular components – Cells or tissue must be rapidly and efficiently disrupted disrupted – Inactivate RNases – Denature nucleic acid-protein complexes – RNA selectively partitioned from DNA and protein protein

8. Ribonucleases (RNases) RNases are naturally occurring enzymes that RNases are naturally occurring enzymes that degrade RNA Common laboratory contaminant (from Common laboratory contaminant (from bacterial and human sources) Also released from cellular compartments Also released from cellular compartments during isolation of RNA from biological samples Can be difficult to inactivate Can be difficult to inactivate

9. Protecting against RNase Wear gloves at all times Wear gloves at all times Use RNase-free tubes and pipet tips Use RNase-free tubes and pipet tips Use dedicated, RNase-free, chemicals Use dedicated, RNase-free, chemicals Pre-treat materials with extended heat (180oC for Pre-treat materials with extended heat (180oC for several hours), wash with DEPC-treated water, NaOH or H2O2 Supplement reactions with RNase inhibitors Supplement reactions with RNase inhibitors Include a chaotropic agent (guanidine) in the procedure Include a chaotropic agent (guanidine) in the procedure – Chaotropic agents such as guanidine inactivate and precipitate RNases and other proteins

10. Samples  RNA could be isolated from:  Tissues.  Cultured cells ( in Monolayer or in Suspension) including human, animal, plant, yeast or bacterial cell.  Body fluids including urine, CSF, ascitic fluid, semen ….etc.  Whole blood.

11. After harvesting or excision, samples can be immediately flash frozen in liquid nitrogen and stored at –70°C. Frozen tissue should not be allowed to thaw during handling or weighing, but cell pellets can partially thaw enough to allow them to be dislodged by flicking. The relevant procedures should be carried out as quickly as possible. Samples can also be stored at –70°C in lysis buffer after disruption and homogenization. Frozen samples are stable for months. Tissues and cells Sample collection, storage, and handling Tissues and cells

12. Cells Cultured cells are lysed and homogenized directly in the lysis buffer. Tissues Tissues can be disrupted using Rotor–stator homogenizers, the TissueLyser system, Mortar and Pestle.

13. Disruption and homogenization using rotor – stator homogenizers In the presence of lysis buffer, rotor–stator homogenizers : disrupt and simultaneously homogenize tissues in 5– 90 s, depending on the toughness of the sample.

14. Disruption and homogenization using the TissueLyser system In bead-milling, tissues can be disrupted by rapid agitation in the presence of beads and lysis buffer. Disruption and simultaneous homogenization occur by the shearing and crushing action of the beads as they collide with the cells.

15. Disruption using a mortar and pestle  Freeze the sample immediately in liquid nitrogen, and grind to a fine powder under liquid nitrogen.  Allow the liquid nitrogen to evaporate, but do not allow the sample to thaw.  Add lysis buffer, and continue as quickly as possible with homogenization.  Transfer the suspension (tissue powder and lysis buffer) into an RNase-free, liquid-nitrogen–cooled polypropylene tube.

16. Whole blood should be collected in the presence of an anticoagulant, EDTA, although other anticoagulants such as citrate, heparin, or ACD (acid citrate dextrose) can also be used. For optimal results, blood samples should be processed within half an hour of collection. To ensure that the isolated RNA contains a representative distribution of mRNAs, blood samples should not be stored before isolating RNA. Blood

17. Blood Blood cells are lysed in two separate procedures, erythrocyte lysis and leukocyte lysis. Erythrocytes (red blood cells) of human blood do not contain nuclei and are therefore not important for RNA isolation since they neither synthesize nor contain RNA. The target of RNA isolation from whole blood is leukocytes (white blood cells), which are nucleated and therefore do contain RNA.

18.  Erythrocyte lysis by hypotonic buffer.  Intact leukocytes are then recovered by centrifugation.  Leukocyte lysis by denaturing buffer that immediately, inactivate RNases, allowing isolation of intact RNA.  Disruption of cells and homogenization of lysate.  Spin columns, unique tools allow fast and simple homogenization of cell lysates without risk of cross- contamination.  Cell lysates are loaded onto the shreder spin column, briefly microcentrifuged, and the homogenized lysate collected in a tube.

19. RNA Extraction Protocols  RNA isolation by Salt precipitation  Organic extraction of total RNA  Affinity purification using spin columns  Selective mRNA isolation

20. Organic Extraction of total RNA Lyse/homogenize cells using trizol reagent/ chloroform and centrifuge. Organic phase separates from aqueous phase Organic solvents on bottom Aqueous phase on top (contains total RNA) Cellular debris and genomic DNA appears as a “ film ” of debris at the interface of the two solutions Remove RNA solution to a clean tube; precipitate RNA and wash with ethanol, then resuspend RNA in water Aqueous Phase Organic solvents

21. Organic Extraction of total RNA Advantages Advantages  Compatible with a variety of sample types  Can process small and large samples  Established and proven technology  Inexpensive Disadvantages Disadvantages  Organic solvents  Not high-throughput  RNA may contain contaminating genomic DNA

22. Affinity purification of total RNA Lyse cells, and spin to remove large particulates/cell debris Apply lysate (containing nucleic acids and cellular contaminants) to column with glass membrane Wash with alcohol to remove contaminants; nucleic acids stick to glass membrane while contaminants wash through. Treat with DNase enzyme to remove contaminating DNA. Apply water to the column; purified RNA washes off the glass and is collected

23. Affinity purification of total RNA Advantages – Eliminates need for organic solvents – Compatible with a variety of sample types (tissue, tissue culture cells, white blood cells, plant cells, bacteria, yeast, etc.) – DNase treatment eliminates contaminating genomic DNA genomic DNA – Excellent RNA purity and integrity

24. Estimating RNA quality by spectrophotometry RNA sample absorbances are determined on the spectrophotometer at 260nm, 280nm, and 230nm – 260nm: nucleic acid (DNA, RNA, nucleotides) – 280nm: protein – 230nm: guanidine

25. Quantitation of RNA Nucleic acids absorb UV light maximally at Nucleic acids absorb UV light maximally at260nm For RNA: For RNA: 1 OD260 Unit = 40 μg/ml of ssRNA The concentration in the sample is calculated The concentration in the sample is calculated by using the formula: A260 x dilution x 40 = [RNA] μg/ml

26. Estimating RNA purity by spectrophotometry A260/A280 – pure RNA will exhibit an A260/A280 ratio within the range of 1.8 - 2.0 – If the RNA exhibits a ratio lower than 1.7, this indicates protein contamination in your sample A260/A230 A260/A230 – properly purified RNA should exhibit an A260/A230 ratio within the range of 1.8 - 2.0. – If the RNA exhibits a ratio lower than 1.7, this indicates guanidine contamination in your sample While these readings give an estimation of purity, they do NOT indicate RNA integrity While these readings give an estimation of purity, they do NOT indicate RNA integrity

27. Evaluating RNA integrity Two major ribosomal subunits: 28S and 18S Approximately 80% of total RNA is associated with these two subunits Approximately 80% of total RNA is associated with these two subunits Integrity of the total RNA sample can be determined by evaluating the 28S (5 kb) and 18S (2 kb) rRNA Integrity of the total RNA sample can be determined by evaluating the 28S (5 kb) and 18S (2 kb) rRNA – 28S and 18S rRNA should appear as distinct bands – Ratio of 28S:18S should be approximately 2:1

28. Protocol of RNA Isolation from Human Peripheral Blood   This protocol assumes that each 4 ml of human blood yield approximately 15 ug of RNA.   RNA is very easily degraded by ever- present RNAses. Therefore, all of the tubes and solutions in this protocol must be RNAse-free.

29. Preparation of white blood cell pellet 1) Add 4 ml fresh human blood in polypropylene conical centrifuge tube. 2) Bring volume to 25 ml with RBC Lysis Buffer. 3) Let stand at room temperature for 10 minutes. 4) Pellet cells at 600 x g (approx 1,400 rpm) for 10 minutes in a 4° C centrifuge. 5) Carefully decant supernatant. 6) Gently resuspend the pellet in 1 ml of RBC Lysis Buffer and transfer to a 1.5 mlmicrocentrifuge tube. Let stand for 2 minutes.

30. 7) Pellet cells for 2 minutes by centrifuging in a microfuge at 3000 rpm. This does not need to be cold. 8) Carefully aspirate the supernatant. 9) If the resulting pellet is still red, repeat the RBC lysis (steps 6, 7, and 8) as needed. 10) Process cells for RNA if desired (or store cells at –80° C until needed).

31. Homogenization 1) 1)Add 800 ul of TRIzol/RNAstat60 solution to each tube and resuspend the cells. 2) 2)Homogenize and lyse the cells. Incubate for 10 minutes on ice. Phase Sepraration 1) 1)Add 0.2 ml of Chloroform (CHCl3) and vigorously shake or vortex each tube for 15 seconds. Let samples stand on ice for 10 minutes. 2) 2)Centrifuge the samples at 17,000 rpm for 15 minutes at 4°C.

32. RNA Precipitation 1) 1)Remove the upper phase and transfer to a clean microcentrifuge tube to which an equivalent volume of ice-cold isopropanol is added. Be careful not to remove any of the white interface when collecting the upper phase of the extraction. 2) 2)Immediately vortex and place on ice for 10 minutes. Note: This is an excellent stopping point. If desired, the samples can be placed in a -20°C freezer overnight instead of the 15 minutes. 3) 3)Samples are centrifuged at 17,000 rpm for 10 minutes at 4°C. Note: you may be able to see a small white pellet of RNA at the bottom of the tube after this step.

33. RNA Wash 1) Carefully decant the supernatant, and rinse the pellet with 0.5 ml of ice-cold 75%ethanol. 2) Centrifuge the samples at 13000 rpm for 8 minutes at 4°C. 3) Decant the supernatant. Wash again. 4) Quick-spin the samples (14,000 rpm, 30 seconds, 4°C) to drive the remaining fluid to the bottom of the tube. 5) Using a P10 pipettor, carefully remove all of the remaining liquid in the bottom of the tube. 6) Allow the pellet to dry for 5 to 10 minutes to remove any remaining ethanol.

34. RNA Solubilization Dissolve the RNA pellet by adding 30 to 50 μl of RNAse-free H2O to each sample. Quantitate (OD 260) and use RNA as needed.

35. Video 2 RNA extraction Yahoo! Video Detail for RNA Isolation workshop.flv Yahoo! Video Detail for RNA Isolation workshop.flv