Recommended Procedures for RNA Extraction from FFPE Tissue. Ghasemi Golestan University of Medcal Science June 2014

Transcription

1 Recommended Procedures for RNA Extraction from FFPE Tissue Ghasemi Golestan University of Medcal Science June 2014

2 RNA Extraction Isolates RNA from other cellular components in the sample Removes inhibitory substances may not eliminate all RNases are ubiquous enzymes which degrade RNA Proper handling and use of RNase-free materials will eliminates degradation of RNA and introduction of RNases

3 Cont Inactivates endogenous RNases in specimen (guanidine isothiocyanate in lysis solution) Storage of isolated RNA: Store in RNase free solution(depc Treated Water) 24 hr. - Store at 4 ৹ C >24 hr. - Store at -70 ৹ C Obtaining high quality RNA

4 RNase Contamination Body fluids such as perspiration gloves tips and tubes Lab surfaces and environment Use certified RNase free tips and tubes(perchase or manual) Dust, bacteria, spores, etc (sterilize ) Water and buffer Use RNase-free water Endogenous RNA Proper handling & storage of specimens

5 Sample types Blood samples Tissue samples Fresh tissue FFPE tissue ( Formalin fixed paraffin embedded tissue) cultured samples Yeast and fungi Plant samples

6 Methods of RNA Extraction Bead-based RNA-isolation procedure Column-based extraction kit Manual procedure withtrizol

7 Manual RNA Extraction Steps Paraffin removal Tissue digestion RNA Extraction

8 Paraffin removal Each sample Cuts by separated sterile blade in a separated plastic plates 1000 μl of xylene to the labeled tube containing your specimen Shaking for 10 S AND spin Centrifuge for 3 minutes ( x g, 22 ৹ C) Withdraw the xylene supernatant Repeat xylene wash steps FOR 3 times Repeat these 3 steps by ethanol 100% Air-dry the pellet for 5 minutes

9 NOTES The size of the used tissue Cutting with microtome(2-5 piece) Cutting with Punch (10-20mg) Check weight with microtupe Crushing The use of chemical hood Paraffin-free tissue Absolutely, dry The time can be changed

10 Tissue Digestion Digestion with Proteinase- K(20.2mg/ml, FERMENTAS) 10µl Proteinase-K + 190µl buffer Proteinase-K =200µl for each specimen Sealing by parafilm Incubate at 56 ৹ C for 3-5hr(Thermo Block) Microfuge

11 NOTES PH of buffer = 8 Pipetting and vortex after you add a buffer & proteinase- k Temp for RNA 56 ৹ C Time is variable( 3 hr - over night) Vortex of samples per min Almost complete dissolving(sometimes with additional proteinase- k) The buffer storage in room temperature Maintenance of the sample at -20 ৹ C to 24 hours

12 Total RNA Extraction TRIZOL, Ready to use reagent for the isolation of total RNA Mono-phasic solution of phenol and guanidine isothiocyanate Sample Lysis With Trizol Reagent Cell Lysis Sample: homogenized tissue Disruption of cells and Dissolving of cell components

13 Notes Laminar hood Except for the Trizol step(chemical hood) Uv for 10 min Use certified RNase free tools and water Sterilization Trizol to be kept away from light and oxidation and storage at 4 ৹ C Stages of RNA purification on ice

14 Trizol Extraction protocol Tissue Homogenize with Trizol (1ml,SIGMA) Chloroform(0.2ml) is added for phase separation allowing collection of the aqueous phase containing RNA( µl) RNA is precipitated with addition of Isopropyl(0.5ml) Incubation at -20 ৹ C for 1 hour Final wash with ethanol(75%)

15 RNA Pellet Briefly dry pellet for 5-10 min Do not let pellet dry completely or over-dry as this will decrease solubility Reconstitute pellet with RNase-free water(30µl) Vortex reconstitute pellet prior to pipetting Separating some of the samples(4µl) to check quality and quantity of RNA

16 Measuring the RNA by picodrop OD خلوص 260/280(RNA )

17 Notes TRIZOL will be gradually added to homogenizing Homogenize with pellet mixer, 5cc syringe, Incubation stage is variable: Room temp 20min Freezer -20 ৹ C for 1hr Freezer -20 ৹ C overnight Maintenance of obtained RNA at freezer -70 ৹ C

18 Notes cont Generally not be done the gel electrophoresis for FFPE tissue, because: Eukaryotic (5.8S : 156 nt, 28S : 5070 nt,18s : 1869 nt)>100bp Treatment with DNase1: after digestion or extraction Isopropyl would be added = volume of collected aqueous phase The quality of the RNA from paraffin embedded tissues will be much lower than fresh tissues

19 Why is it so difficult to isolate good quality RNA from FFPE tissues? Severe degradation of the RNA by The process of fixing the tissue sample and embedding it Cross-linkage between nucleic acids and proteins Covalent modification of RNA by mono-methylol (-CH2OH) addition to the bases

20 RNA Extraction Set up Weight of samples Increase of Incubation in xylene Ph of buffer Proteinase- K Time of incubation in proteinase-k Trizol and Homogenize Isopropyl Removal of the supernatant phase Time of incubation in freezer -20

21 Good Luck