LCM Protocols – RNA handling
Contents
1. N Carl Zeiss Microscopy LCM Protocols RNA handling We make it visible LCM Protocols RNA handling Non contact Laser Capture Microdissection Carl Zeiss Microscopy Germany Content O XO XO tO o iO OY Ul O UBD BWW O M0 ON NNN OD N UI UI A AA WUWUDNDDDNDDND DO O 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 Suggested UV treatment Poly L Lysine treatment Mounting samples onto slides Frozen sections Formalin Fixed Paraffin Embedded FFPE sections Cytospins Blood and tissue smear Staining procedures Formalin Fixed 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 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 miRNA from frozen sections RNA from FFPE sections miRNA from FFPE sections Using other extraction methods One sample for DNA and RNA Quality control of RNA General remarks on RNA distribution content RNase activ2. Vries i Iran rruacugnocbad nei a drum c adem ji 1 i f FISH Immunofluorescence Proteins PALM User Protocols FISH Hybridization Imriunofluorescence on frozen sections barr Bee iira em EH Fiona li ae Pa niku No BID For questions comments or information requests please contact ZEISS Microscopy Labs Application Lab labs zeiss de ooo 49 8990 9000900 o 27 LCM Protocols RNA handling For scientific questions please contact labs zeiss de 49 8990 9000 900 www zeiss de applab February 2012 Carl Zeiss Microscopy GmbH 07745 Jena Germany BioSciences 49 8990 9000 800 palm info zeiss de i l We make it visible www zeiss de microdissection
3. 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 we strongly 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 Ambion 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 e g BBC Biochemical 3900 4 stain 10 seconds in EOSIN Y e g SIGMA HT110 2 32 5 perform a quick increasing ethanol series 7096 96 100 6 air dry shortly Storage Stained slides can be used immediately or stored at 80 C 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
4. between dot and 0 the sample and acts as a stabilizing backbone during lifting Therefore even large areas Due to the short working distance of the are lifted by a single laser pulse without affec 100x or 150x magnitying objectives only ting the morphological integrity 0 17 mm thin cover glass slides can be used Use of Membraneslide is especially important for isolating single cells chromosomes as well MembraneSlide NF nuclease free is certified as live cells or small organisms to be free of DNase RNase and human DNA Carl Zeiss Microscopy offers slides 1 mm In addition to PEN MembraneSlide ZEISS 0 17 mm covered with polyethylene naph also offers polyethylene teraphthalate PET thalate PEN membrane This PEN membrane membrane covered slides These slides are is highly absorptive in the UV A range which recommended for fluorescence applications facilitates laser cutting due to a better signal to noise ratio The membrane can be used for all kind of applications Alternatively the PET membrane is available When working with low magnifying objec attached to a metal frame FrameSlide PET tives like 5x or 10x both regular 1 mm thick This frame structure is resistant to microwave glass slides and 0 17 mm glass slides can be treatment The slide should be submerged used To keep this flexibility for higher magni completely in buffer for heating The special fications 20x 40x or 63x ZEISS recommends bonding is inert and adapted to heat
5. more details and handling please see Two different microfuge tube sizes with Liquid Cover Glass product information these filled caps are available from ZEISS For more details and handling please see AdhesiveCap product information AdhesiveCap opaque Order No 415190 9201 000 500 ul AdhesiveCap opaque Order No 415190 9181 000 200 ul Liquid Cover Glass Order No 415190 9020 000 LCM Protocols RNA Handling Collection devices AdhesiveCap Other microfuge tubes The intention of AdhesiveCap is to allow Other commercially available RNase free plas LCM Laser Capture Microdissection with ticware 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 possible RNase activities 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 bi RNases from regular tubes lization there is no risk of evaporation and crystal formation during extended speci Treatment of microfuge tubes men harvesting to remove RNases AdhesiveCap is produced with cleanroom add 0 1 ml DEPC to 100 ml of double technology and can therefore be consi distilled water to get a 0 196 DEPC dered as RNase free Autoclaving is not solution DEPC e g ROTH K028 1 possible ZEISS recommends AdhesiveCap as a coll 1 stir for 5 6 h at room temperature to ection device fo
6. procedure can help additionally against RNA degradation or PCR inhibition 12 This short staining procedure colors the nuclei violet and the cytoplasm weak violet Mucin mastcells amyloid or developing bone are stained red It is recommended for RNase rich tissues since all solutions contain high ethanol concentrations Procedure 1 after fixation 2 min 70 ethanol dip slide into 1 Cresyl Violet Acetate solution for 30 sec 2 remove excess stain on absorbent surface 3 dip into 7096 ethanol 4 dip into 10096 ethanol 5 air dry shortly 1 2 min Dissolve solid Cresyl Violet Acetate e g ALDRICH 86 098 0 at a con centration of 196 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 LCM 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 5096 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
7. sequentially by differential solubilization of the same pre cious FFPE sample Pure DNA and RNA are obtained from the entire sample ZEISS Microscopy Labs have successfully tested this kit for its applicability for LCM samples on several FFPE materials Both DNA and RNA can efficiently be purified simultaneously from LCM samples when some modifications to the original ma nual AllPrep DNA RNA FFPE Handbook 09 2010 are made The first Proteinase K digestion and lysis step before separating the DNA and RNA fractions seems critical especially for the RNA yield Mostly we have found a time of one hour instead of 15 minutes to be a good com promise for both DNA and RNA yield but this may vary depending on the individual material We strongly encourage perfor ming pilot experiments with time courses of the first digestion time between one to three hours For more information please contact ZEISS Microscopy Labs directly since an universal protocol recommendation is presently not available Note Proteinase K digestion time should be optimized for any tissue sample At least 1 hour at 56 C is recommen ded in the first lysis step of the QIAGEN AllPrep amp DNA RNA Kit but longer times may be more efficient Due to the normally very small sample size from LCM a direct quality control of the RNA requires high sensitivity technologies Gel electrophoresis or even NanoDrop procedures are frequently not sensitive enough The Agile
8. treat using long distance objectives ment up to 95 C so that the membrane does not ruffle during the heating process With those objectives you have the possibility If you need more information about these to adapt the working distance to the different slides please contact glass slides by moving the correction collar on the objective labs zeissde MembraneSlide 1 0 MembraneSlide 1 0 MembraneSlide 0 17 PEI MembraneSlide 50x1 0 P MembraneSlide 1 0 PET FrameSlide PET LCM Protocols RNA Handling Preparation of slides Treatment to remove RNases Samples on glass slides With PALM MicroBeam almost every kind of biological material can be microdissected and lifted directly from glass slides using the AutoLPC function of PALM RoboSoft ware 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 wate
9. 0 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 LCM Protocols RNA Handling Downstream Applications 20 miRNA from frozen sections To capture microdissected samples from frozen sections ZEISS Microscopy Labs recommend AdhesiveCap 500 yl The special miRNeasy Mini Kit QIAGEN 217004 combined with AdhesiveCap in our hands results in very good yield and quality of total RNA including enrichment of small RNAs Only minor adaptations have to be made to the original QIAGEN procedure see miRNeasy Mini Handbook 10 2007 when applying AdhesiveCap Quality control by direct analysis like the Agilent Bioanalyzer RNA 6000 Pico Lab Chip 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 uim thickness Modifications for LCM samples As first step add 350 ul of QIAzol Lysis Reagent to the tube of the AdhesiveCap instead of the usual 700 ul due to the smaller tube size Close cap and invert the tube The lysis should be performed upside down for 30 minutes at room temp
10. a will interfere with laser efficiency Removing this medium is done by dipping the slide 5 6 times into ice cold RNase free water If staining is done in aqueous solutions the supporting substance is 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 it is helpful to let the slides dry overnight in a drying oven at 56 C The longer melting drying step will strongly improve the adhesion of the section to the membrane 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 strongly inhibiting cutting and lifting If you are working with un stained sections it is therefore important to remove the paraffin completely before laser cutting and lifting If applying standard staining procedures deparaffination is routinely included in any protocol 1 mm MembranesSlides 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 LCM 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 materi
11. act 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 Proteinase K Procedure 1 Add 20 ul digestion buffer containing Proteinase K 150 mM NaCl 100 mM Tris pH 7 5 0 596 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 Do not use any waterbath 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 appropriate lysis buffer and mix by intense vortexing if not proceeding immediately store the digested samples a 20 9g See 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 23 LCM Protocols RNA Handling Downstream Applications One sample for DNA and RNA Quality control of RNA 24 The QIAGEN AllPrep amp DNA RNA Kit 880234 is designed for simultaneous purification of genomic DNA and total RNA including small RNAs from FFPE material DNA and RNA are released
12. al 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 10096 methanol brane which would lead to leakage Allow the cytospins to dry at room tempe during fixation or washing steps and rature before staining therefore would impair the LMC process Let smears air dry shortly and fix them for 2 up to 5 minutes in 7096 ethanol LCM 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 Cresyl Violet FFPE sections After deparaffination continue with the staining procedure of your choice Most standard staining procedures can be used for FFPE sections for recommen dations see Frozen sections Frozen sections Most standard histological stainings e Q HE Methyl Green Cresyl Violet Nuclear Fast Red are compatible with subsequent RNA isolation Note Using frozen sections endogenous RNases may still be active after the short fixation step Therefore it is recommeded to keep all incubation steps as short as possible Please use RNase free water and solutions for all steps All required reagents should be kept on ice At ZEISS Microscopy Labs we usually perform the Cresyl Violet or Hematoxylin staining Skipping the Eosin staining of the HE
13. asy MinElute spin column is 2 ul elution with 14 ul of RNase free water results in a 12 ul eluate 15 The RNA solution may be stored at 20 C or used directly for reverse transcription LCM Protocols RNA Handling miRNA from FFPE sections In contrast to MRNA and rRNA miRNAs are mostly well preserved in FFPE tissue and this allows quite good yields from LCM samples Due to their small size the miRNAs are nor mally not bound efficiently by the QIAGEN columns during the regular FFPE Kit proce dure Still the miRNA fraction can be strongly enriched by simply changing one washing step of the standard protocol To capture microdissected samples from FFPE sections ZEISS Microscopy Labs recommend AdhesiveCap Modifications for miRNA samples 1 Collection and extraction is initially done according to our protocol for total RNA from FFPE see page 22 applying components of the QIAGEN RNeasy FFPE Kit Order No 73504 Step 1 till step 7 are performed as usual 2 Atstep 8 an increased volume of 1120 ul ethanol 100 is added to the tube instead of the normal 720 ul With this stronger dilution the binding conditions change in favour of small RNAs Therefore miRNAs are no longer lost in the flow through 3 All further steps of the routine protocol step 9 12 remain unchanged and can be done as usual Using other extraction methods Apart from the QIAGEN Kits there are many other possibilities and kits to extr
14. at 28000 x g 210000 rpm Discard the flow through Reuse the collection tube in step 10 10 Repeat step 9 until the entire sample has passed through the RNeasy MinElute spin column Reuse the collection tube in step 11 11 Add 500 ul Buffer RPE to the RNeasy MinElute spin column Close the lid gently and centrifuge for 15 sec at 28000 x g 210000 rpm to wash the spin column membrane Discard the flow through Reuse the collection tube in step 12 Note Buffer RPE is supplied as a concentrate Ensure that ethanol is added to Buffer RPE before use 12 Add 500 ul Buffer RPE to the RNeasy MinElute spin column Close the lid gently and centrifuge for 2 min at 28000 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 13 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 through It is important to dry the spin column membrane since residual ethanol may interfere with downstream reactions 14 Place the RNeasy MinElute spin column in a new 1 5 ml collection tube Add 14 30 yl RNase free water directly to the spin column membrane Close the lid gently and centri fuge for 1 min at full speed to elute the RNA The dead volume of the RNe
15. between successful and unsuccessful experiments DOs designate a special area for working with RNA Clean benches with special cleaning solutions e g RNaseZap AMBION 49780 wear gloves and change them frequently use sterile disposable plasticware glassware should be treated with 0 196 DiEthylPyroCarbonate DEPC or oven baked at 180 C for at least 4 hours before use Use pipette tips with filters aqueous solutions should be treated with 0 196 DEPC use only RNase free reagents tubes and tips 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 Store prepared RNA at 80 C 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 in general use protocols e g staining with short incubation times on ice DON Ts don t touch anything with bare hands dont autoclave pipette tips as water vapor may contain RNases e don t allow frozen tissue to thaw dontresuspend RNA in DEPC water residual DEPC can inhibit downstream reactions LCM Protocols RNA Handling Preparation of slides Samples on MembraneSlide MembranesSlides are glass slides covered with Regular glass slide 1 mm thick 1 a membrane on one side thin slide 0 17 mm thick gt dot This membrane is easily cut together with DuplexDish and FrameSlide
16. erature After short vortexing spin down briefly in a tabletop centrifuge and transfer the liquid to a 1 5 ml reaction tube Add another 350 ul of QlAzol Lysis Reagent and mix by pipetting Add 140 ul Chloroform CCI and close tube securely Shake vigorously or vortex for at least 15 seconds Note After step 3 switch directly to the QIAGEN manual at step 4 Place tube at room temperature for 2 3 min All further steps are now performed according to the normal QIAGEN procedure steps 4 13 The RNA elution in step 13 should be done with the minimal volume 30 ul to avoid unnecessary dilution of the RNA The final RNA solution may be stored at 20 C or used directly for downstream reactions LCM Protocols RNA Handling Downstream Applications RNA from FFPE sections For collecting microdissected samples ZEISS Microscopy Labs recommend AdhesiveCap ZEISS Microscopy Labs apply the QIAGEN RNeasy FFPE Kit 73504 with some LCM specific modifications please see page 22 This procedure is very effective and allows a high final concentration of RNA due to a small elution volume 12 ul Genomic DNA contamination is minimized by a DNase I digest on the purification column Deparaffination and staining is done ac cording to standard procedures for slides please see pages 10 12 and 13 The incubation with Proteinase K in our proto col is prolonged significantly compared to the QIAGEN RNeasy FFPE prot
17. eus 21 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 5 COS7 250 5 Mouse tissue 100 mg Brain 11240 5 Heart 120 6 Intestine 150 2 Kidney 250 5 Liver 400 14 Lung 130 6 Spleen 250 7 Quantitative hierarchy of RNase activity Also the RNase activities vary dramatically across different tissues Krosting J Latham G AMBION Inc A comparison of total RNase activities for 8 different mouse tissues showed that total RNase activity spans in mouse tissues AMBION Inc Fold increase relative to brain Mouse tissues 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 25 26 LCM Protocols RNA Handling ZEISS Microscopy Labs Tips for working with RNA For best RNA quality we use fresh 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 Any condensation of moisture on the section can lead to reactivation of intrinsic RNase activities and therefore fast degradation of RNA A prognosis of the expected amount of RNA is difficult since many factors will influence the outcome see above From mouse liver f
18. g Applying components of the QIAGEN RNeasy FFPE Kit Order No 73504 1 Add 150 ul Buffer PKD and 10 ul of Proteinase K to the tube containing the LCM elements in the AdhesiveCap and invert tube to get contact between liquid and adhesive surface 2 Use an incubator to digest the samples in an upside down position at 56 C overnight or for at least 3 hours then vortex and heat at 80 C for precisely 15 min in a heating block Note Please do not use any water bath for the upside down incubation Incubate on ice for 3 min 4 Add 16 ul DNase Booster Buffer and 10 ul DNase stock solution Mix gently by inverting the tube Centrifuge briefly to collect residual liquid from the sides of the tube Note DNase is supplied lyophilized and should be reconstituted as described in Preparing DNase I stock solution page 14 RNeasy FFPE handbook 09 2010 DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the tube Do not vortex 5 Incubate at room temperature for 15 min 6 Transfer the lysate to a new 1 5 ml microcentrifuge tube 7 Add 320 ul RBC to adjust binding conditions and mix the lysate thoroughly 8 Add 720 ul ethanol 100 to the sample and mix well by pipetting Do not centrifuge Proceed immediately to step 9 9 Transfer 700 ul of the sample to a RNeasy MinElute spin column placed in a 2 ml collection tube Close the lid gently and centrifuge for 15 sec
19. he solution 100yl all over the membrane area e g with a soft hair brush or a pipet tip moved horizontally along the surface Let air dry at room temperature for 2 3 minutes Avoid any injury of the membra ne as this might result in impairment of Laser Capture Microdissection LCM LCM Protocols RNA Handling Mounting samples onto slides Frozen sections Formalin Fixed Paraffin Embedded FFPE sections Sectioning Sectioning 10 Sections are mounted onto Membrane Slides similarly as when using glass slides by melting the frozen section to the warmer slide Freezing media like OCT or similar may be used but should be kept to a minimum and must be removed before laser cutting To our experience massive steel knives al low better section quality than the exchan geable razor blades in the microtome 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 the section from the knife by touching with the warmed area and dry at 20 C in the cryostat for 2 3 minutes Longer drying can increase the adhesion of weakly attaching tissue sections Fixation As first step the dehydration in ice cold 7096 ethanol for 2 3 minutes is always recommended Removing the tissue freezing medium If OCT or another tissue freezing medium is used it is important to remove it before laser cutting because such medi
20. ity ZEISS Microscopy Labs Tips for working with RNA Other protocols DNA Chromosomes Live Cells Introduction Some remarks on RNA 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 LCM 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
21. le with large microdissected samples about 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 LCM Protocols RNA Handling Applying components of the QIAGEN RNeasy Micro Kit Order No 74004 1 Add 350 ul Buffer RLT containing Mercaptoethanol to the tube with the LCM elements in the AdhesiveCap close the cap and incubate in an upside down position for 30 min Please do not use any water bath for the incubation Thorough lysis is essential for good RNA yield Note Mercaptoethanol ME must be added to Buffer RLT before use Add 10 ul S 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 purified 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 35
22. ll areas will stick onto the wet inner surface of the cap and will not fall down after the lifting procedure Be aware that aqueous solu tions will dry out after a while When using glass mounted samples and AutoLPC filling the cap with liquid comple tely can increase the capturing efficiency 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 LD 20x LD 40x and LD 63x objectives 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 LCM Protocols RNA Handling Downstream Applications RNA from frozen sections To capture microdissected samples from frozen sections ZEISS Microscopy Labs recommend AdhesiveCap For RNA extraction a procedure of choice can be used The RNeasy Micro Kit QIAGEN 74004 combined with AdhesiveCap 500 ul in our hands results in very good yield and quality of RNA from various tissues 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 Lab Chip Kit is limited to concentrations above 50 pg ul and may only be possib
23. nt 2100 Bioanalyzer RNA 6000 Pico LabChip Kit is able to analyze RNA samples with concentrations down to 50 pg ul and provides information about RNA quality degradation purity and quantity see also www chem agilent com A similar technology is available from BioRad Experion System see also www bio rad com A prognosis of the expected amount of RNA in FFPE tissue is very difficult since many factors like species cell tissue type fixa tion staining fragmentation extraction procedure and others will influence the outcome Anyway RNA from FFPE material will frequently not show clear profiles and only rough estimations of the size distribution and the RNA amount are possible LCM 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 1 596 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 much as 396 of the mRNA pool whereas others account for less then 0 0196 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 nucl
24. ocol because all our tests with laser microdissected mate rial from various tissues showed higher RNA yields when 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 Quality control by direct analysis like the Agi lent Bioanalyzer RNA 6000 Pico LabChip Kit is very limited and may only be possible with quite large microdissected samples of ten some A mm collected area from tissue sections of 5 10 um thickness We normally 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 oligo dT as primers for the cDNA synthesis Note The use of random or gene specitic primers is important Reverse transcription of formalin fixed RNA with standard oligo dT primers is inefficient and strongly 3 pri me biased due to the numerous strand breaks and modifications inflicted by the formalin fixation and paraffin embedding procedure 21 22 LCM Protocols RNA Handlin
25. r 30 50 C 2 time needed for the coverslip to swim off may range from hours to days 3 gentle movement 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 MembranesSlides are shipped without any pretreatment To ensure RNase free MembranesSlides or glass slides use dry heat at 180 C for 4 hours to completely inactivate RNases 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 Suggested UV treatment To overcome the hydrophobic nature of the membrane we strongly recommend 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 slides with Poly L Ly sine 0 1 w v e g SIGMA P8920 may help for poorly adhering materials e g brain sections and should be done after UV treatment Distribute a drop of t
26. r all RNA experiments dissolve the DEPC 2 soak the reaction tubes into the DEPC For more details and handling please see solution take care that the tubes are also AdhesiveCap product information completely covered with liquid not blistered and incubate overnight at room temperature 3 autoclave the tubes together with me TE RTT 892 OM USATE 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 toxic and should be used under a hood AdhesiveCap opaque Order No 415190 9201 000 500 ul AdhesiveCap opaque Order No 415190 9181 000 200 ul AdhesiveCap clear Order No 415190 9211 000 500 ul AdhesiveCap clear Order No 415190 9191 000 200 ul 16 Collection procedures Dry collection procedure Please have a look into the PALM MicroBeam User Manual Dry collection AdhesiveCap AdhesiveCap is the recommended collec tion 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 Note Please do not use any water bath for the upside down incubation Subsequently briefly centrifuge the lysate and then apply the routine RNA extraction procedure Wet collection other microfuge tubes Pipette 20 ul lysis buffer into the cap The lifted cells or ce
27. rozen 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 Archival tissues are mostly Formalin Fixed and Paraffin Embedded RNA extraction from these tissues is often not very effective because of the crosslinking 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 microdissection Summarized recommendations Keep attention to DOs and DON Ts on handling RNA page 6 e Take AdhesiveCap as collection device for all RNA experiments page 16 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 73504 results in good RNA yield quality and quantity from FFPE tissue in our lab page 21 LCM Protocols RNA Handling Brochures and Protocols Live cells Chromosomes DNA uaran umm bam aei jue mian em dari Ja PALM User Protocols Laser Micromeanipulation in Life Exciesrecomaa s Chromosome Preparation
28. the container should not be opened before it is completely warmed up again to ambient temperature LCM 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 results in impaired view of morphology Diffusor Holders for PALM RoboMover and PALM CapMover Il are equipped with diffusors The opaque glass diffuses the incident mi croscope 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 Tube collector 2x200 CMII Order No CollectorSet SingleTube 200 RM Order No 415101 2000 410 415101 2000 951 LCM 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 due smoothens the rough tissue surface resulting to enhanced color balance and contrast in enhanced morphology which makes the view comparable to those of coverslipped tissue sections For
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