Purification of total RNA, including miRNA, from
Contents
1. 2015 PreAnalytix all rights reserved A PreAnalytiX A QIAGEN BD Company www PreAnalytiX com2. Carrier RNA The PAXgene Tissue miRNA Kit contains poly A RNA for use as carrier RNA When added to lysates from microdissected tissue the carrier RNA may improve the recovery of total RNA including miRNA Carrier RNA is not required when processing more than 5000 cells The small amounts of poly A RNA used as carrier RNA do not interfere with subsequent RT PCR even when oligo dT is used as a primer for reverse transcription Reverse transcription reactions typically contain an excess of oligo dT primers and the small amounts of poly A used as carrier RNA are insignificant in comparison Total RNA purified using poly A RNA as carrier RNA can be amplified with the QIAGEN QuantiTect Whole Transcriptome Kit which uses a mix of random and oligo dT primers miRNA purified using poly A RNA as carrier RNA can be amplified with the QIAGEN miScript PCR System Starting material Starting material for total RNA including miRNA purification is a PFPE or PFCE tissue section mounted on an glass adhesion slide for manual microdissection procedure A or on a frame slide with a membrane for laser microdissection procedure B Sections must be prepared according to the Supplementary Protocol Preparation of sections from PAXgene Tissue fixed paraffin embedded PFPE and PAXgene Tissue fixed cryo embedded PFCE tissue for manual or Laser microdissection LMD Detach the microdissected specimen from the slide and transfer it to a dedicated collection devi
3. 1 before use Add 10 ul B ME per 1 ml Buffer TM1 Dispense in a fume hood and wear appropriate protective clothing Buffer TM1 containing B ME can be stored at room temperature 15 25 C for up to 1 month Pre cool the Buffer TM1 on ice If using the RNase Free DNase Set for the first time prepare a DNase stock solution Dissolve the solid DNase in 550 ul DNase resuspension buffer RNase free water provided with the set Do not vortex the reconstituted DNase DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the tube Reconstituted DNase can be stored at 2 8 C for up to 6 weeks and at 15 C to 30 C for up to 6 months When processing lt 5000 cells carrier RNA may be added to the lysate see Carrier RNA page 2 Before using carrier RNA for the first time dissolve it 310 ug in 1 ml RNase free water Store this stock solution at 20 C and use it to make fresh dilutions for each set of RNA preparations The concentration of this stock solution is 310 ng pl Prepare a working solution of 4 ng ul using Buffer TMI Procedure A Purification of total RNA including miRNA from manually microdissected PFPE and PFCE tissue In the procedure below refers to PFPE tissue and A refers to PFCE tissue 1 Label the lid and the body of a 1 5 ml safelock microcentrifuge tube not provided Prepare a lysis mixture in the tube by pipetting 75 pul RNase free water and
4. 10 pl Proteinase K Mix by gently flicking the tube and centrifuge briefly 1 2 s at 500 1000 x g to collect residual liquid from the sides of the tube Note When processing lt 5000 cells add 20 ng of carrier RNA 5 ul of a 4 ng ul solution to the mixture Prepare a dilution of carrier RNA as described in Things to do before starting Total RNA including miRNA from microdissected PFPE PFCE tissue PX19 Oct 15 page 3 of 1 10 11 Using a microtome or A cryostat make a tissue section of 6 12 um thickness from a paraffin embedded or A cryo embedded tissue block Capture the tissue section on an RNase free adhesion slide Remove embedding medium and stain optional according to the Supplementary Protocol Preparation of sections from PAXgene Tissue fixed paraffin embedded PFPE and PAXgene Tissue fixed cryo embedded PFCE tissue for manual or laser microdissection LMD Keep freshly prepared slides in ice cold ethanol until microdissection For manual microdissection remove the slide from the ethanol Using an absorbent sheet wipe away the liquid on the slide that surrounds the tissue section Note Tissue areas of interest can be isolated by scratching away other tissue parts with a scalpel blade or cell scraper Remove tissue portions from the slide immediately after taking the section out of the ethanol Avoid completely drying the section because this makes it harder to dissolve the tissue from the sl
5. PreAnalytiX Supplementary Protocol Purification of total RNA including miRNA from microdissected PAXgene Tissue fixed paraffin embedded PFPE and PAXgene Tissue fixed cryo embedded PFCE tissues This protocol is for using the PAXgene Tissue miRNA Kit to purify total RNA including miRNA from PAXgene Tissue fixed paraffin embedded PFPE and PAXgene Tissue fixed cryo embedded PFCE tissues that have been manually or laser microdissected LMD from a slide IMPORTANT The tissue samples must be fixed and stabilized in PAXgene Tissue Containers The PAXgene Tissue Container Product Circular includes information on tissue fixation and stabilization For instructions on the preparation of PFCE tissue blocks read the PreAnalytiX Supplementary Protocol Cryo embedding tissue specimens fixed and stabilized with the PAXgene Tissue System PX14 To prepare sections from PFPE and PFCE tissue blocks for microdissection follow instructions of the Supplementary Protocol Preparation of sections from PAXgene Tissue fixed paraffin embedded PFPE and PAXgene Tissue fixed cryo embedded PFCE tissue for manual or laser microdissection PX20 Please read the PAXgene Tissue miRNA Kit Handbook paying careful attention to the Safety Information and Important Notes sections before beginning this procedure For Research Use Only Not for use in diagnostic procedures No claim or representation is intended to provide information fo
6. ce for direct purification of total RNA including miRNA IMPORTANT The tissue source the preanalytical workflow and the choice of materials used to make a PFPE and PFCE specimen significantly affect quality and quantity of isolated RNA Furthermore variability in quality and quantity of isolated RNA may be observed across species and tissue type Make sure that instruments have been checked and calibrated according to the manufacturer s recommendations t This is not a complete list of suppliers and does not include many important vendors of biological supplies Total RNA including miRNA from microdissected PFPE PFCE tissue PX19 Oct 15 page 2 of 1 Things to do before starting If working with RNA for the first time read Appendix A General Remarks on Handling RNA in the PAXgene Tissue miRNA Kit Handbook Prepare 80 ethanol by mixing ethanol 96 100 purity grade p a and RNase free water Set the temperature of the shaker incubator to 45 C for PFPE tissues or to 56 C for PFCE tissues Buffer TM1 and Buffer TM2 both concentrate and working solution may form a precipitate during storage If necessary warm to 37 C to dissolve Buffer TM2 and Buffer TM3 are supplied as concentrates Before using the buffers for the first time add 3 volumes of isopropanol to Buffer TM2 and 4 volumes of ethanol 96 100 purity grade p a to Buffer TM3 to obtain working solutions Add B Mercaptoethanol B ME to Buffer TM
7. ide Place the slide on a_ horizontal working plate and overlay the section with 85 pl ice cold Buffer TM1 Make sure that the whole section is covered Note Work quickly We recommend using a dark underlay as a work surface to make it easier to see the tissue The volume of Buffer TM1 needed depends on the tissue surface area Detach the tissue on the slide by pipetting the lysis mixture up and down Transfer the tissue and all liquid to the labeled 1 5 ml safelock microcentrifuge tube from step 1 and mix by vortexing for 5 s Note If the tissue does not dissolve easily use the pipet tip to scrape the tissue from the slide Incubate the tissue on a shaker incubator for 15 min at 1400 rpm and 45 C or A 56 C After incubation centrifuge briefly 1 2 s at 500 1000 x g to remove drops from the tube lid Add 240 ul ethanol 96 100 purity grade p a Mix by vortexing for 5 s and centrifuge briefly 1 2 s at 500 1000 x g to remove drops from the tube lid Pipet the sample including any precipitate that may have formed into a PAXgene RNA MinElute spin column red positioned in a 2 ml processing tube Centrifuge for 15 s at 8000 x g Place the spin column in a new 2 ml processing tube and discard the old processing tube containing the flow through Continue with step 15 of the protocol Purification of Total RNA Including miRNA from Sections of PFPE Tissue in the PAXgene Tissue miRNA Kit Handbook page 19 Note The elut
8. ion volume can vary between 14 40 ul and strongly depends on the area and number of microdissected cells Low input requires lower elution volumes for high concentrations of RNA Furthermore repeated elution steps increase the overall total RNA yield of a sample Total RNA including miRNA from microdissected PFPE PFCE tissue PX19 Oct 15 page 4 of 1 Procedure B Purification of total RNA including miRNA from laser microdissected LMD PFPE and PFCE tissue 1 Using a microtome or A cryostat make a tissue section of 6 12 um thickness from a paraffin embedded or A cryo embedded tissue block Capture the tissue section on a frame slide with membrane for laser microdissection 2 Remove embedding medium and stain optional according to the Supplementary Protocol Preparation of sections from PAXgene Tissue fixed paraffin embedded PFPE and PAXgene Tissue fixed cryo embedded PFCE tissue for manual or laser microdissection LMD Keep freshly prepared slides in ice cold ethanol until laser microdissection 3 Using a Laser Microdissection System dissect a tissue area of gt 5000 um gt 50 cells from the PFPE or PFCE LMD slide Note lt 50 cells may require pre amplification of cDNA for downstream applications use for example the QIAGEN REPLI g WTA Single Cell Kit 4 Collect cells directly into a dedicated collection tube filled with 15 ul Buffer TM1 use for example the cap of a 0 5 ml PCR tube when using a Leica LMD
9. new 2 ml processing tube and discard the old processing tube containing the flow through Total RNA including miRNA from microdissected PFPE PFCE tissue PX19 Oct 15 page 5 of 1 11 Continue with the wash of step 15 of the protocol Purification of Total RNA including miRNA from sections of PFPE tissue in the PAXgene Tissue miRNA Kit Handbook page 19 Note The elution volume can vary between 14 40 ul and strongly depends on the area and number of microdissected cells Low input requires lower elution volumes for high concentrations of RNA Furthermore repeated elution steps increase the overall total RNA yield of a sample For up to date licensing information and product specific disclaimers see the respective PreAnalytiX or QIAGEN kit handbook or user manual Handbooks and user manuals are available at www qiagen com and www preanalytix com or can be requested from QIAGEN Technical Services or your local distributor Safety data sheets SDS for any QIAGEN or PreAnalytiX product can be downloaded from www qiagen com safety Trademarks PAXgene PreAnalytiX PreAnalytiX GmbH QIAGEN QuantiTect REPLI g QIAGEN Group CORNING AXYGEN Corning Inc Eppendorf Eppendorf AG Leica Leica Microsystems IR GmbH SuperFrost VWR VWR International LLC Registered names trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law PX19
10. r the diagnosis prevention or treatment of a disease Equipment and reagents When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate safety data sheets SDSs available from the product supplier e RNase free water e Ethanol 96 100 purity grade p a e Xylene e 14 3 M B mercaptoethanol B ME commercially available solutions are usually 14 3 M e Variable speed microcentrifuge capable of attaining 1000 8000 x g and equipped with a rotor for 2 ml microcentrifuge tubes e Microtome or cryostat Make sure that instruments have been checked and calibrated according to the manufacturer s recommendations PreAnalytiX 0 A QIAGEN BD Company www PreAnalytiX com e Shaker incubator capable of incubating at 45 80 C and shaking at 400 rpm not exceeding 1400 rpm e g Eppendorf Thermomixer Compact t e Vortex mixer e Adhesion slides for manual microdissection e g SuperFrost Plus Slides VWR Cat no 631 0108 or frame slides for laser microdissection e g PPS membrane 25mm x 76 mm Leica Cat no 11505273 t e Laser Microdissection System e g Leica LMD6500 amp LMD7000 Leica Microsystems e 1 5 ml safelock microcentrifuge tubes e 0 5 ml PCR tubes suitable for the dedicated LMD system e g CORNING AXYGEN 0 5 ml Tube with Flat Cap Cat no 10169 8901 for Leica LMD6500 amp LMD7000
11. system Note If not possible add Buffer TM1 immediately after collecting the cells Make sure to add B ME to Buffer TM1 before use see Things to do before starting 5 Add 60 pl Buffer TM1 Mix by vortexing for 30 s and centrifuge briefly 1 2 s at 500 1000 x g to remove drops from the tube lid Note The volume of Buffer TM1 depends on the collection vessel used for microdissection but should not exceed 80 ul Lysates in Buffer TM1 can be frozen at 80 C however freezing may impact RNA yield when a low number of cells is used Thaw frozen lysates on ice and proceed with step 6 6 If processing lt 5000 cells 20 ng carrier RNA 5 pl of a 4 ng pl solution may be added to the lysate Prepare the carrier RNA as described in Things to do before starting 7 Add 75 pl RNase free water and 10 pl Proteinase K and mix by vortexing for 5 s Note Do not mix Buffer TM1 and Proteinase K before adding them to the sample 8 Incubate on a shaker incubator for 15 min at 45 C or A 56 C and 1400 rpm After incubation centrifuge briefly 1 2 s at 500 1000 x g to remove drops from the tube lid 9 Add 240 ul ethanol Mix by vortexing for 5 s and centrifuge briefly 1 2 s at 500 1000 x g to remove drops from the tube lid 10 Pipet the sample including any precipitate that may have formed into a PAXgene RNA MinElute spin column red positioned in a 2 ml processing tube Centrifuge for 15 s at 8000 x g Place the spin column in a
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