Sample staining, mounting, and cutting for RNA extraction
Contents
1. By using the software function go to check point the slide is moved out of the light path and the cap can be lowered further towards the objectives for looking inside 17 18 PALM Protocols RNA Handling Downstream Applications RNA from frozen sections For RNA extraction a procedure of choice can be used To capture microdissected samples from frozen sections ZEISS Labs recommend AdhesiveCap The RNeasy Micro Kit QIAGEN 74004 combined with AdhesiveCap 500 ul in our hands results in very good yield and quality of RNA For recommended modifications to the original QIAGEN protocol please see page 19 The final RNA solution 12 ul may be stored at 20 C or used directly for reverse trans cription Quality control by direct analysis like the Agilent Bioanalyzer RNA 6000 Pico LabChip Kit is limited to concentrations above 50 pg ul and may only be possible with large microdissected samples some 2 mm of collected areas from tissue sections of 5 10 um thickness We normally use 5 to 10 ul of the final RNA solution as template in a RT reaction of 20 ul e g Transcriptor First Strand cDNA Synthesis Kit ROCHE 04 379 012 001 PALM Protocols RNA Handling Applying the components of the QIAGEN RNeasy Micro Kit 1 Add 350 ul Buffer RLT containing Mercaptoethanol to the tube with the LCM elements in the AdhesiveCap then vortex and incubate in an upside down position for 30 min2. N Carl Zeiss Microlmaging PALM Protocols RNA handling We make it visible PALM Protocols RNA handling Non contact Laser Capture Microdissection Carl Zeiss Microlmaging Location Munich Germany Content VOOVDOnnoau Introduction Some remarks on RNA The DOs and DON Ts on handling RNA Preparation of slides Samples on Membraneslide Samples on glass slides Archived samples removing the coverslip Treatment to remove RNases UV treatment Poly L Lysine treatment Mounting samples onto slides Frozen sections Paraffin embedded FFPE sections Cytospins Blood and tissue smear Staining procedures Paraffin embedded FFPE sections Frozen sections Cresyl Violet Hematoxylin Eosin HE Storage Non contact Laser Capture Microdissection LCM Procedures Tips to improve morphological information Diffusor CM AdhesiveCap opaque LiquidCover Glass Collection devices AdhesiveCap Other microfuge tubes Collection procedures Dry collection AdhesiveCap Wet collection other microfuge tubes Capture check looking into the cap to see the lifted samples Downstream Applications RNA from frozen sections RNA from FFPE sections Using components of the QIAGEN RNeasy FFPE Kit Using other extraction methods Quality control of RNA General remarks on RNA distribution content RNase activity LabTips for working with RNA Other protocols DNA Chromosomes Live Cells Introduction Some remarks on RNA
3. All required reagents should be kept on ice At ZEISS Labs we usually perform Dissolve solid cresyl violet acetate the Cresyl Violet or Hematoxylin Eosin HE e g ALDRICH 86 098 0 at a con staining centration of 1 w v in 50 EtOH at room temperature with agitation stirring for several hours to overnight Filter the staining solution before use to remove unsolubilized powder Sometimes Lot to Lot variations in the purchased cresyl violet powder can lead to weaker staining results if the dye content is below 75 PALM Protocols RNA Handling Note In most cases this cresyl violet staining procedure will be sufficient for cell identification If an enhancement of the staining is desired a reinforcement by two additional steps in 50 ethanol first before the staining in cresyl violet second after the staining in cresyl violet is possible Additional intensification can be obtained by increasing the working temperature of all solutions to room temperature The endogenous RNase acitivity varies between different tissues please see page 23 Therefore when the short staining protocol is modified by additional steps 50 ethanol or by increasing the working temperature ZEISS Labs stron gly recommend a quality control of the RNA please see page 22 Ambion offers the LCM Staining Kit 1935 which also contains a cresyl violet dye When using this kit we recommend to omit the final xylene step of the Am
4. Cover Glass Order No 415190 9020 000 PALM Protocols RNA Handling Collection devices AdhesiveCap Other microfuge tubes The intention of AdhesiveCap is to allow Other commercially available RNase free LCM Laser Capture Microdissection with plasticware can be used too out applying any capturing liquid into the e g ABgene AB 0350 0 5 ml tubes caps prior to LCM This minimizes RNase activity If there are no RNase free tubes available Beside the quick relocation of the lifted use the following procedure to remove samples in the cap due to instant immo RNases from regular tubes bilization there is no danger of evaporation and crystal formation during extended Treatment of microfuge tubes specimen harvesting to remove RNases i ks details and Pale please see SHOT TIP onoo mie double also AdhesiveCap product information distilled water to get a 0 1 DEPC solution DEPC e g ROTH K028 1 Note CZMI recommends AdhesiveCap as a collection device for all RNA experiments 1 stir for 5 6 h at room temperature to dissolve the DEPC 2 soak the reaction tubes into the DEPC solution take care that the tubes are completely covered with liquid not blistered and incubate overnight at room temperature 3 autoclave the tubes together with the solution for 20 minutes at 121 C to inactivate the DEPC 4 discard the liquid carefully and thoroughly Dry the tubes at 50 C 80 C 5 use the tubes as usual Note DEPC is toxi
5. RNA is a biological macromolecule with many different functions Messenger RNA mRNA transcribed from DNA serves as template for synthesis of proteins This protein synthesis is carried out by ribosomes which consist of ribosomal RNA rRNA and proteins Amino acids for protein synthesis are delivered to the ribosome on transfer RNA tRNA molecules RNAs are also part of riboproteins and ribozymes Analysis of RNA can provide a good reflection of an organism s gene expression profile Gene expression profiling of material isolated by microdissection has become a very important method for analyzing cellular behavior in a micro scale and is used in research and clinical applications Therefore the isolation of high quality RNA is crucial for all subsequent steps and the success of the overall experiment PALM Protocols RNA Handling The DOs and DON Ts of handling RNA RNA degradation is a common reason for failing experiments RNA is prone to digestion by a wide variety of endogenous and exogenous RNases These RNases are present on almost any object that comes into contact with human skin and are difficult to inactivate Even minute amounts are sufficient to destroy RNA Some precautions can make the difference between an intact and degraded RNA prep see also www ambion com and therefore between successful and unsuccessful experiments DOs e designate a special area for working with RNA e clean benches with special cleaning s
6. sections are stai ned in aqueous solutions the supporting substance is normally removed automa tically by the water containing steps Sections are mounted onto MembraneSlides the same way as routinely done using glass slides Floating the section on warm water as well as hot plate techniques can be applied After mounting let dry the slides overnight in a drying oven at 56 C To allow laser cutting and lifting a coverslip and standard mounting medium must not be applied Deparaffination Paraffin will reduce the efficiency of the laser sometimes completely inhibiting cutting and lifting If you are working with unstained sections it is therefore very important to remove the paraffin before laser cutting and lifting If applying standard staining procedures deparaffination is routinely included in any protocol 1 mm MembraneSlides can be used like normal glass slides Minimal procedure 1 Xylene 2 minutes 2 times 2 Ethanol 100 1 minute 3 Ethanol 96 1 minute 4 Ethanol 70 1 minute PALM Protocols RNA Handling Cytospins Blood and tissue smear Cytospins can be prepared on glass slides Distribute a drop of peripheral blood or on MembraneSlides After centrifugation or material of a smear over the slide with a cytocentrifuge let the cells air dry Be careful to avoid injuries in the mem Then fix for 5 minutes in 100 methanol brane which would lead to leakage Allow the cytospins to dry at room tempe during fix
7. Thorough lysis is essential for good RNA yield Note Mercaptoethanol ME must be added to Buffer RLT before use Add 10 ul ME per 1 ml Buffer RLT Dispense in a fume hood and wear appropriate protective clothing Buffer RLT is stable at room temperature for 1 month after addition of ME 2 Spin down the lysate in a microcentrifuge for 5 minutes 13400 rcf e g Eppendorf 5415D 12000 rpm Note Samples can now be stored for later use at 80 C or extracted immediately following the original protocol of the QIAGEN RNeasy Micro Kit Handbook 04 2003 3 To continue with the isolation transfer the lysate to a RNase free 1 5 ml microcentrifuge tube 4 Now switch to step 5 of the QIAGEN protocol Total RNA Isolation from Microdissected Cryosections RNeasy Micro Handbook 04 2003 pp 20 5 Add 1 volume 350 ul of 70 ethanol to the homogenized lysate and mix well by pipetting Do not centrifuge Continue immediately with step 6 Note All further steps 6 14 of the QIAGEN protocol remain unchanged and should be performed step by step as listed there Please consider also the comments and tips of the QIAGEN RNeasy manual especially the section Things to do before starting PALM Protocols RNA Handling Downstream Applications RNA from FFPE sections 20 For collecting microdissected samples ZEISS Labs recommend AdhesiveCap ZEISS Labs prefer the QIAGEN RNeasy FFPE Kit 74404 with som
8. ample and acts as a stabilizing backbone during lifting Therefore even large areas are lifted by a single laser impulse without affecting the morphological integrity Use of MembraneSlide is especially important for isolating single cells chromosomes as well as live cells or small organisms Carl Zeiss Microlmaging CZMI offers slides 1 mm 0 17 mm covered with polyethylene naphthalate PEN membrane This PEN membrane is highly absorptive in the UV A range which facilitates laser cutting The membrane can be used for all kind of applications When working with low magnifying objec tives like 5x or 10x both regular 1 mm thick glass slides and 0 17 mm glass slides can be used To keep this flexibility for higher magni fications 20x 40x or 63x CZMI recommends using long distance objectives With those objectives you have the possibility to adapt the working distance to the different glass slides by moving the correction collar on the objective Due to the short working distance of the 100x magnifying objectives only 0 17 mm thin cover glass slides can be used MembraneSlide NF nuclease free is certified to be free of DNase RNase and human DNA In addition to PEN MembraneSlide CZMI also offers polyethylene teraphthalate PET membrane covered slides These slides are helpful for special processes i e for isolation of chromosomes and some fluorescence applications Alternatively the PET membrane is available atta
9. appropriate lysis buffer and mix by intense vortexing if not proceeding immediately store the digested samples ait 20 C or 20C 6 Continue with your preferred extraction procedure Note Proteinase K digestion time should be optimized for any tissue sample at least 3 hours are recommended but up to 18 hours may be more efficient The most common method used for assessing the integrity of total RNA is to analyze the RNA sample on an agarose gel In general at least 200 ng of RNA must be loaded onto the gel To analyze RNA samples with concentrations down to 50 pg ul the Agilent 2100 Bioana lyzer is an alternative to traditional gel based analysis and provides information about RNA quality degradation purity and quan tity see also www chem agilent com A prognosis of the expected amount of RNA in a tissue is difficult since many factors like species cell tissue type fixation staining fragmentation extraction proce dure and others will influence the outcome PALM Protocols RNA Handling General remarks on RNA distribution content RNase activity A typical mammalian cell contains 10 30 pg total RNA mRNA rRNA tRNA The majority of RNA molecules are tRNAs and rRNAs mRNA represents only 1 5 of the total cellular RNA Approximately 360 000 mRNA molecules are present in a single cell corresponding to approximately 12 000 different transcripts with a typical length of 2 kb Some mRNAs comprise as mu
10. ation or washing steps and rature before staining therefore would impair the laser capture microdissection process Let smears air dry shortly and fix them for 2 up to 5 minutes in 70 ethanol PALM Protocols RNA Handling Staining procedures For isolation of high quality RNA use only freshly prepared and precooled staining solutions and take notice of our tips on handling RNA please see page 24 Formalin Fixed Paraffin Embedded FFPE sections Cresyl Violet After deparaffination continue with the This short staining procedure colors the staining procedure of your choice nuclei violet and the cytoplasm weak violet Most standard staining procedures can It is recommended for RNase rich tissues be used for FFPE sections for recommen since all solutions contain high ethanol dations see Frozen sections concentrations Frozen sections 1 after fixation 2 min 70 Ethanol dip slide for 30 sec into 1 cresyl violet Most standard histological stainings e g acetate solution HE Methyl Green Cresyl Violet Nuclear Fast Red are compatible with subsequent 2 remove excess stain on absorbent RNA isolation surface 3 dip into 70 Ethanol Note Using frozen sections endogenous gt 77 RNases may still be active after the short Zen fixation step Therefore it is recommeded to 5 air dry shortly 1 2 min keep all incubation steps as short as possible Please use RNase free water and solutions for all steps
11. bion instruction manual because xylene makes the tissue very brittle and reduces the ad hesion of the section to the PEN membrane Hematoxylin Eosin HE HE staining is used routinely in most histo logical laboratories and does not interfere with good RNA preparation if intrinsic RNase activity is low The nuclei are stained blue the cytoplasm pink red Procedure 1 after fixation quickly dip slide 5 6 times in RNase free distilled water 2 stain 1 2 minutes in Mayer s Hema toxylin solution e g SIGMA MHS 32 3 rinse 1 minute in DEPC treated tap water or blueing solution BBC 3900 4 stain 10 seconds in Eosin Y e g SIGMA HT1 10 2 32 5 perform a quick increasing ethanol series 70 96 100 6 air dry shortly Storage Stained slides can be used immediately or stored at 80 C before LCM To avoid excess condensation of moisture during thawing the slides should be frozen in a tightly sealed container e g two slides back to back in a 50 ml Falcon tube For rethawing the container should not be opened before it is completely warmed up again to ambient temperature PALM Protocols RNA Handling Non contact Laser Capture Microdissection LCM Procedures Please additionally have a look into the PALM MicroBeam user manual Tips to improve morphological information Embedding and glass covering of the specimen is inapplicable for LCM Thus the rough open surface of the section material often res
12. c and should be used under a hood AdhesiveCap opaque Order No 415190 9201 000 500 pl AdhesiveCap opaque Order No 415190 9181 000 200 pl AdhesiveCap clear Order No 415190 9211 000 500 pl AdhesiveCap clear Order No 415190 9191 000 200 pl 16 Collection procedures Dry collection procedure Please have a look into the PALM MicroBeam user manual Dry collection AdhesiveCap Note CZMI recommends AdhesiveCap as collection device for all RNA experiments Capturing without liquid minimizes RNase activity After LCM add a lysis buffer of your own choice e g QIAGEN 350 ul RLT buffer and incubate upside down for 30 minutes Subsequently centrifuge the lysate and then apply the routine RNA extraction procedure Note Please do not use any water bath for the upside down incubation Wet collection other microfuge tubes Pipette 20 ul lysis buffer into the cap The lifted cells or cell areas will stick onto the wet inner surface of the cap and will not fall down after the lifting procedure Be aware that aqueous solutions will dry out after a while When using glass mounted samples it may be advisory to put more liquid up to 40 ul into the cap Capture check looking into the cap to see the lifted samples To control the efficiency of lifting it is possible to have a look into the collection device e g microfuge cap with the 5x 10x 20x 40x and 63x objectives
13. ch as 3 of the mRNA pool whereas others account for less then 0 01 QIAGEN Bench guide RNA distribution in a typical mammalian cell Total RNA per cell 10 30 pg 80 85 rRNA 28S 18S 5S 15 20 tRNAs snRNAs low MW species 1 5 mRNAs Total RNA in nucleus 14 DNA RNA in nucleus 2 1 mRNA molecules 2x10 1x10 per cell Typical mRNA size 1900 nt RNA content in various cells and tissues TotalRNA mRNA ug ug Cell cultures 107 cells NIH 3T3 120 3 HeLa 150 3 COS7 350 5 Mouse tissue 100 mg Brain 120 5 Heart 120 6 Intestine 150 2 Kidney 350 9 Liver 400 14 Lung 130 6 Spleen 250 7 Quantitative hierarchy of RNase activity Also the RNase activities vary dramatically across in mouse tissues AMBION Inc different tissues Krosting J Latham G AMBION Inc Mousetissues Eaidiincreace A comparison of total RNase activities for 8 different relative to brain mouse tissues showed that total RNase activity spans Pancreas 181 000 a 181 000 fold range from pancreas to brain which Spleen 10 600 points out the importance of RNase control Lung 5 300 Liver 64 Thymus 16 Kidney 8 Heart 2 Brain 1 23 24 PALM Protocols RNA Handling ZEISS Labs Tips for working with RNA For best RNA quality we use frozen sections on MembraneSlides Frozen sections should not be stored for more than a few days at 80 C After staining and drying freezing should be performed in an air tight container A prognosi
14. ched to a metal frame FrameSlide PET In fluorescence applications FISH even weak signals can be detected due to low signal to noise ratio The frame structure of FrameSlide PET is resistant to microwave treatment The special bonding is inert and adapted to heat treatment up to 95 C so that the membrane does not ruffle during the heating process If you need information about these slides please contact E Mail labs zeiss de FrameSlide PE PALM Protocols RNA Handling Preparation of slides Samples on glass slides With PALM MicroBeam almost every kind of biological material can be microdissected and lifted directly from glass slides Even archival pathological sections can be used after removing the cover slip and the mounting medium To facilitate easy lifting additional adhesive substances or Superfrost charged slides should only be applied when absolutely necessary for the attachment of poorly adhering material e g some brain sections or blood vessel rings In those cases higher laser energy is needed for lifting Archived samples removing the coverslip Depending on the applied mounting medium whether it is soluble in xylene or water the whole slide should be completely submerged in the respective solvent 1 standing up in a glass jar filled with either pure xylene or warm water 30 50 C 2 time needed for the coverslip to swim off may range from hours to days 3 gentle moveme
15. e specific modifica tions This procedure is very effective and allows a high final concentration of RNA due to a small elution volume Genomic DNA con tamination is minimized by a special DNA removal column gDNA Eliminator spin column Since normally only stained tissue sections are used for microdissection the deparaffi nation and staining is done according to standard procedures for slides please see pages 10 12 and 13 Furthermore the incubation with Proteinase K in PALM protocols is prolonged significantly compared to the QIAGEN RNeasy FFPE protocol because all our tests with laser microdissected material from various tissues showed higher RNA yields applying longer digestion times Note For formalin fixed samples a Pro teinase K digestion step is essential The time necessary for optimal Proteinase K digestion depends on many factors like tissue type fixation procedure or element size of lifted material An overnight digestion 12 18 hours is a good starting point for optimization but shorter digestion times may be tested as well To our experience at least 3 hours digestion should be applied with any extraction procedure and material The RNA solution may be stored at 20 C or used directly for reverse transcription Quality control by direct analyses like the Agilent Bioanalyzer is very limited and only possible with large microdissected samples some 4 mm from tissue sections of 5 10 um thickness We n
16. ed to elute the RNA The dead volume of the RNeasy MinElute spin column is 2 ul elution with 14 ul of RNase free water results in a 12 ul eluate The RNA solution may be stored at 20 C or used directly for reverse transcription Note Quality control by direct analysis like the Agilent Bioanalyzer Pico chip is very limited and may only be possible with quite large microdissected samples often some 4 mm collected area from tissue sections of 5 10 um thickness 21 PALM Protocols RNA Handling Downstream Applications Using other extraction methods Quality control of RNA 22 Apart from the QIAGEN Kit there are many other possibilities and kits to extract RNA from FFPE material Depending on the material and the experience of the user even simple procedures like homemade AGTC methods or Trizol can be quite efficient If the original extraction protocol does not contain any Proteinase K digestion step we recommend to apply a simple procedure as listed below Procedure 1 Add 20 ul digestion buffer containing Proteinase K 150 mM NaCl 100 mM Tris pH 7 5 0 5 Igepal 0 5 ug ul Proteinase K to the tube containing the LCM elements in the AdhesiveCap 2 Use an incubator to digest the samples in an upside down position at 55 C over night 3 Spin down the lysate in a microcentrifuge 13400 rcf e g Eppendorf 5415D 12000 rpm 4 Inactivate Proteinase K by heating to 90 C for 10 minutes 5 Add the
17. ge 20 PALM Protocols RNA Handling Brochures and protocols Live cells Chromosomes DNA Issachen trom cari zess Weweieseren tan C PALM User Protocols Laser Micromanipulation ji in Life Sciences Chromosome Preparation FISH Immunofluorescence RNA PALM User Protocols FISH Hybridization Immunofluorescence on frozen sections For questions comments or protocol requests please contact ZEISS Labs E Mail labs zeiss de Hotline 49 8990 9000 900 25 NNI PALM Protocols RNA handling For scientific questions please contact E Mail labs zeiss de Hotline 49 8990 9000 900 www zeiss de labs August 2011 Carl Zeiss Microlmaging GmbH 07740 Jena Germany BioSciences Location Munich Phone 49 8990 9000 800 Telefax 49 8990 9000 820 E Mail palm info zeiss de www zeiss de microdissection We make it visible
18. ly adhering materials e g brain sections and should be performed after UV treatment Distribute a drop of the solution on top of the slide Let air dry at room temperature for 2 3 minutes Avoid any leakage of the mem brane as this might result in impairment of Laser Capture Microdissection PALM Protocols RNA Handling Mounting samples onto slides Frozen sections Formalin Fixed Paraffin Embedded FFPE sections Sectioning Sectioning 10 Sections are mounted onto Membrane Slides the same way as routinely done using glass slides To allow subsequent cutting and lifting a coverslip and standard mounting medium must not be applied Freezing media like OCT or similar may be used but should be kept to a minimum and have to be removed before laser cutting For optimal RNA protection take a pre cooled slide and touch the backside of the slide with your finger gloves to warm only the region for placing the section Now transfer section from the knife by touching with the warmed area and dry at 20 C in the cryostat for 2 3 minutes Fixation CZMI recommends the dehydration in ice cold 70 ethanol for 2 3 minutes Removing the tissue freezing medium If OCT or another tissue freezing medium is used it is important to remove it before Laser Microdissection because these media will interfere with laser efficiency Removing the medium is easily done by dipping the slide 5 6 times in ice cold RNase free water If the
19. nt of the jar may speed up the process 4 air dry the slide after removal Note It is very important NOT to use any force to push off the coverslip because this might damage the section Wait till it falls off by itself The necessary time depends on the age of the sample and the dryness of the mounting medium Fresh slides only days old can be decover slipped much faster From the dry glass slide sample material can be lifted directly by AutoLPC function of PALM RoboSoftware Treatment to remove RNases Treatment of MembraneSlides and glass slides to remove RNases are identical Slides are shipped without any pretreat ment e To ensure RNase free MembraneSlides or glass slides heat slides at 180 C for 4 hours to completely inactivate RNases e MembraneSlide NF nuclease free is certified to be free of DNase RNase and human DNA Treatments to remove nucleases are there fore not necessary using these slides UV treatment To overcome the hydrophobic nature of the membrane it is advisable to irradiate with UV light at 254 nm for 30 minutes e g in a cell culture hood The membrane gets more hydrophilic therefore the sections paraffin and cryosections adhere better Positive side effects are sterilization and destruction of potentially contaminating nucleic acids Poly L Lysine treatment Additional coating of the slide with Poly L Lysine 0 1 w v e g SIGMA P8920 only will be necessary for poor
20. olutions e g RNaseZap AMBION 9780 e wear gloves and change them frequently e use sterile disposable plasticware e glassware should be treated with 0 1 DiEthylPyroCarbonate DEPC or oven baked at 180 C for at least 4 hours before use e use pipette tips with filters aqueous solutions should be treated with 0 1 DEPC e use only RNase free reagents tubes and tips e for best results use samples that have been snap frozen on dry ice or in liquid nitrogen all required reagents should be kept on ice e store prepared RNA aliquoted in ethanol or RNA elution buffer at 80 C e to avoid condensation of moisture during thawing the slides should be frozen at 80 C and rethawed in a tightly sealed container e g 50 ml Falcon tube e in general use protocols e g staining with short incubation times on ice DON Ts e don t breath on samples some researchers wear masks e don t touch anything with bare hands e don t autoclave pipette tips as water vapor may contain RNases e don t allow frozen tissue to thaw e don t resuspend RNA in DEPC water residual DEPC can inhibit downstream reactions PALM Protocols RNA Handling Preparation of slides Samples on MembraneSlide MembraneSlides are special slides covered with a membrane on one side This membrane is easily cut together with Regular glass slide 1 mm thick gt 1 thin slide 0 17 mm thick gt dot DuplexDish and FrameSlide gt between dot and 0 the s
21. ormally use 5 to 10 ul of the final RNA solution in a RT reaction of 20 ul e g Transcriptor First Strand cDNA Synthesis Kit ROCHE 04 379 012 001 using random oligomers instead of oligoT as primers for the cDNA synthesis Note The use of random or gene specific primers is important Reverse transcription of formalin fixed RNA with standard oligoT primers is inefficient and strongly 3 prime biased due to the numerous strand breaks and modifications inflicted by the formalin fixation and paraffin embedding procedure PALM Protocols RNA Handling Using components of the QIAGEN RNeasy FFPE Kit 1 Add 150 ul Buffer PKD and 10 ul of Proteinase K to the tube containing the LCM elements in the AdhesiveCap and vortex in an upside down position Use an incubator to digest the samples in an upside down position at 55 C overnight or for at least 3 hours then vortex and heat at 80 C for 15 min in a heating block Add 320 ul of Buffer RBC to adjust binding conditions Mix the lysate thoroughly and transfer it to a gDNA Eliminator spin column placed in a 2 ml collection tube Centrifuge for 30 sec at gt 8000 x g e g Eppendorf 5415D gt 10000 rpm Discard the column and save the flow through Add 720 ul of 100 ethanol to the flow through and mix well by pipetting Do not centrifuge Proceed immediately to the next step Transfer 700 ul of the sample to a RNeasy MinElute spin column placed in a 2 ml collec
22. s of the expected amount of RNA is difficult since many factors will influence the out come see above From mouse liver frozen sections we usually are able to retrieve 5 20 pg RNA per cell calculated from extractions of 1000 cells and analysis with an Agilent Bioanalyzer Agilent Application Note 5988 EN on our website or at www chem agilent com Note Quantitative results from an analysis with the Agilent Bioanalyzer RNA Pico kit are dependent on the salt content of the sample Archival tissues are mostly formalin fixed and paraffin embedded RNA extraction from these tissues is not very effective because of the cross linking properties of aldehydes Other methodologies for preservation of high molecular weight RNA in FFPE tissue are described by Vincek et al 2005 Diagn Mol Pathol 14 3 127 133 and Olert et al 2001 Pathol Res Pract 197 823 826 For more information see our website www zeiss de publications Summarized recommendations e Keep attention to DOs and DON Ts on handling RNA page 6 e Take AdhesiveCap as collection device for all RNA experiments page 16 e Choose a short staining procedure for tissues with high content of endogenous RNases e g Cresyl Violet page 12 e RNeasy Micro Kit QIAGEN 74004 results in good RNA yield quality and quantity from frozen sections in our lab page 18 e RNeasy FFPE Kit QIAGEN 74404 results in good RNA yield quality and quantity from FFPE tissue in our lab pa
23. tion tube Close the lid gently and centrifuge for 15 sec at gt 8000 x g 210000 rpm Discard the flow through Reuse the collection tube in step 7 Repeat step 6 until the entire sample has passed through the RNeasy MinElute spin column Reuse the collection tube in step 8 Add 500 ul Buffer RPE to the RNeasy MinElute spin column Close the lid gently and centrifuge for 15 sec at gt 8000 x g 10000 rpm to wash the spin column membrane Discard the flow through Reuse the collection tube in step 9 Note Buffer RPE is supplied as a concentrate Ensure that ethanol is added to Buffer RPE before use Add 500 ul Buffer RPE to the RNeasy MinElute spin column Close the lid gently and centrifuge for 15 sec at gt 8000 x g 210000 rpm to wash the spin column After centrifugation carefully remove the spin column from the collection tube so that the column does not contact the flow through Place the RNeasy MinElute spin column in a new 2 ml collection tube and discard the old collection tube with the flow through Open the lid of the spin column and centrifuge at full speed for 5 min Discard the collection tube with the flow througn It is important to dry the spin column membrane since residual ethanol may interfere with downstream reactions Place the RNeasy MinElute spin column in a new 1 5 ml collection tube Add 14 30 ul RNase free water directly to the spin column membrane Close the lid gently and centrifuge for 1 min at full spe
24. ults in impaired view of morphology Diffusor CM Diffusor CM can be inserted into PALM Cap Mover like any holder and is moved over the sample The opaque glass diffuses the incident microscope light which smoothens the harshness of contrast and depending on material and staining even minute details as nuclei and cell boundaries show up Even slight differences in color become visible For more details and handling please see Diffusor CM product information Holders for PALM RoboMover and PALM CapMover Il are equipped with diffusors PALM CombiSystem Diffusor CM Order No 415101 2100 320 14 PALM Protocols RNA Handling AdhesiveCap opaque Liquid Cover Glass The white opaque filling of AdhesiveCap The polymeric and low viscose Liquid Cover clearly improves visualization of morpho Glass completely embeds the tissue and logical information of the samples at the smoothens the rough tissue surface resulting object plane due to enhanced color ba in enhanced morphology lance and contrast which makes the view comparable to those of coverslipped tissue For more details and handling please see sections Liquid Cover Glass product information Two different microfuge tube sizes with these filled caps are available from CZMI For more details and handling please see AdhesiveCap product information AdhesiveCap opaque Order No 415190 9201 000 500 pl AdhesiveCap opaque Order No 415190 9181 000 200 pl Liquid
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