Dynabeads® mRNA DIRECT™ Micro Kit User Guide
Contents
1. b Tap the plate to resuspend the beads in the nuclease free water then let sit 30 seconds at room temperature If the solution appear homogenous continue to the next step If not pipet to resuspend the beads before continuing to the next step c Place the plate on the Magnetic Stand 96 After the solution clears transfer the supernatant containing the mRNA to a new tube without disturbing the pellet Store the isolated mRNA on ice for immediate use or store for up to two weeks at 80 C For guidelines on using the isolated mRNA in downstream applications see Appendix B on page 28 Dynabeads mRNA DIRECT Micro Kit User Guide 21 Chapter 3 mRNA Isolation from Purified Total RNA mRNA isolation from gt 1 50 ug total RNA samples Assess the yield and size distribution of the mRNA 22 Assess the yield and size distribution of the mRNA using the appropriate method for your initial sample input Total RNA initial sample input Recommended method 28 ug 1 Quantitate the yield of the mRNA using the Qubit RNA Assay Kit Cat no Q32852 with a Qubit Fluorometer Refer to Qubit RNA Assay Kits Manual Man no MAN0002327 or the Qubit 2 0 Fluorometer User Manual Man no MAN0003231 for instructions Note You can use a NanoDrop Spectrophotometer in place of the Qubit RNA Assay Kit and Qubit Fluorometer For increased accuracy we recommend that you quantitate the RNA concentration using the Q2. 80 C PCR amplification 1 Immediately before adding the reverse transcription PCR mix place the tube containing the Dynabeads mRNA complex on the magnet for 1 minute then discard the supernatant 2 Add reverse transcription PCR reaction mix e For one tube PCR Resuspend the Dynabeads mRNA complex in 50 uL reverse transcription PCR mix and transfer to the PCR tube e For two step PCR Resuspend the Dynabeads mRNA complex in the reverse transcription PCR reaction mix according to the manufacturer s recommendation 3 Perform cDNA synthesis as recommended by the manufacturer of the reverse transcriptase When using a thermostable reverse transcriptase and the bead bound oligo dT as primer for first strand cDNA synthesis an initial incubation at 50 C for 5 minutes is necessary before proceeding at the recommended temperature For additional guidelines on using the isolated mRNA in downstream applications see Appendix B on page 28 Dynabeads mRNA DIRECT Micro Kit User Guide 13 2 Chapter 2 Direct mRNA Isolation from Microscale Samples from Cells or Tissues mRNA isolation from tumor cells mRNA isolation from tumor cells Before you begin Prepare lysate from tumor cells isolated from whole blood or MNC Isolate mRNA for PCR amplification 14 IMPORTANT To prevent RNA degradation by RNase contamination use gloves and change them frequently Dynabeads Epithelial Enrich coated with the monoclo
3. Place the sample tube on the magnet for 1 minute then discard the supernatant Remove the sample tube from the magnet and resuspend the Dynabeads mRNA complex in 100 uL Washing Buffer A by careful pipetting Place the sample tube on the magnet for 1 minute then discard the supernatant Repeat steps 5 6 once Resuspend the Dynabeads mRNA complex in 100 uL Washing Buffer B Transfer the suspension to a new tube Place the new sample tube on the magnet for 1 minute then discard the supernatant Resuspend the Dynabeads mRNA complex in 100 uL Washing Buffer B Place the sample tube on the magnet for 1 minute then discard the supernatant Remove the sample tube from the magnet and resuspend the Dynabeads mRNA complex in 100 uL of ice cold 10 mM Tris HCl Store the sample tube on ice for immediate use in PCR amplification Note We recommend that you immediately use the Dynabeads mRNA complex for reverse transcription If storage is needed elute the mRNA off the beads and freeze the mRNA containing supernatant When storing mRNA it is critical that no RNase is present in your sample If elution of mRNA is necessary add 10 20 uL 10 mM Tris HCl and incubate at 65 C to 80 C for 2 minutes Place the tube on the magnet and immediately transfer the supernatant to a new microcentrifuge tube The eluate may be used immediately for reverse transcription or store for up to two weeks at 80 C Immediately before adding the revers
4. mRNA DIRECT Micro Kit User Guide 15 mRNA Isolation from Purified Total RNA M WOLKE OW iro sesh sie seach She senna ob 0 ni E niece a patina a e adored 16 m Materials and equipment required but not provided 00 17 E mRNA isolation from gt 1 50 ug total RNA samples 000000065 17 E mRNA isolation from 100 ng 1 ug total RNA samples 5 23 Workflow Wash the Dynabeads Oligo dT 25 Prepare the total RNA sample s Perform two rounds of mRNA isolation in the same well Prepare the RNA for binding Round 1 Bind the mRNA to the Dynabeads Wash the mRNA Elute the mRNA Round 2 Re bind the mRNA to the Dynabeads v Wash the mRNA Elute the mRNA 16 Dynabeads mRNA DIRECT Micro Kit User Guide Chapter 3 mRNA Isolation from Purified Total RNA Materials and equipment required but not provided Materials and equipment required but not provided Item Source Cat no Nuclease free water Life Technologies AM9938 Optional Exfold ERCC Spike In Mix Life Technologies 4456739 or or ERCC RNA Spike In Mix 4456740 Magnetic Stand 96 Life Technologies AM10027 or or Magnetic Ring Stand 96 Well AM10050 or or DynaMag 2 Magnet 12321D Pipettors and RNAse free pipette tips Major laboratory suppliers MLS 1 2 mL 96 well U bottom plate MLS Optional Non Stick RNase free Microfuge Life Technologies AM124
5. or see Appendix C Ordering Information on page 30 for a list of appropriate total RNA purification products Quantitate the amount of RNA in the sample using a Qubit Fluorometer or NanoDrop Spectrophotometer 1 Dilute the 100 ng 1 ug total RNA sample in nuclease free water to a final volume of 50 uL IMPORTANT If you will add spike in control mix see next step reduce the final RNA sample volume to allow for the appropriate volume of spike in mix For example for 100 ng Total RNA input bring the sample volume to 48 uL to allow for the addition of 2 uL spike in mix in the next step Dynabeads mRNA DIRECT Micro Kit User Guide 23 3 Chapter 3 mRNA Isolation from Purified Total RNA MRNA isolation from 100 ng 1 ug total RNA samples 2 Recommended for gene expression experiments Add an ERCC Spike In Control Mix to the diluted total RNA sample See the ERCC RNA Spike In Control Mixes User Guide Part no 4455352 for mix selection and sample preparation procedures The following chart indicates the correct dilution of ERCC to be added to the total RNA sample based on input Amount of total RNA Volume of Spike In Mix 1 or Mix 2 dilution t 100 ng 2 uL 1 1000 250 ng 1 uL 1 200 500 ng 1 uL 1 100 750 ng 3 pL 1 200 1 ug 2 uL 1 100 t ERCC RNA Spike In Mix 1 ExFold Spike In Mix 1 or ExFold Spike In Mix 2 Perform two 1 Prepare the RNA for binding rounds of mRNA a Heat the total R
6. tore ENA Dp Dynabeads Oligo dT 25 1 8 ug 30 uL gt 8 20 ug 50 uL gt 20 50 ug 100 uL Place the tube or plate onto the appropriate magnetic stand then allow the suspension to clear Aspirate and discard the clear supernatant without disturbing the beads Remove the tube or plate from the magnetic stand then add an equivalent volume of fresh Lysis Binding Buffer to the beads Vortex to resuspend the beads then quick spin to bring the beads to the bottom of the tube The mixture should appear homogeneous Dynabeads mRNA DIRECT Micro Kit User Guide Chapter 3 mRNA Isolation from Purified Total RNA 3 mRNA isolation from gt 1 50 ug total RNA samples Prepare the total Note Use the highest quality total RNA possible as your starting material Ideally use RNA sample s RNA with an RNA integrity number RIN greater than 7 Use FirstChoice Total RNA for high quality intact RNA isolated from a variety of sources or see Appendix C Ordering Information on page 30 for a list of appropriate total RNA purification products Quantitate the amount of RNA in the sample using a Qubit Fluorometer or NanoDrop Spectrophotometer 1 Dilute the total RNA sample in nuclease free water to bring the sample to the final volume shown in the following table Final RNA sample volume if Final RNA sample volume if UEN IRAKS pul using Spike In Mixt not using Spike In Mix 1 8 ug 150 uL volume Spike In Mix
7. 8 Materials and equipment required but not provided 0 eee eee eee eee eens 9 Prepare reagents and samples 00 000 cece cece eee eee eee ranra 9 Prepare burters occgra eve ew ead dese de hives ee ed ene beta dee ne be tata drag vee tees 9 Wash Dynabeads Oligo dT o5 before use 0 6 cece eee tenn ees 9 Prepare Samples eeraa 3 5 ERAI Petes ENEE tee AEA oe Hades Med aee at 10 mRNA isolation from cultured cells and cell suspensions 00000e cece eee eees 10 Beforeyou begin ics caxageaedecteeeeaed gore ec ee ie ee eee 10 Prepare lysate from cultured cells and cell suspensions 000000eee ee eees 10 Isolate MRNA for PCR amplification 00 000 c cece eee eee eee eens 10 PCER amplification stata ute te fags Pec ah at Ad a se aes deat Seah oe ha to 11 MRNA isolation from tissues 00 e ented ene teen eaae 12 Before youbegimi c 2254 028 aha lialgutes fog bide ee deans Gee aaey SNARE 12 Prepare lysate from plant and animal tissues 00000 c eee e cece eee eee es 12 Isolate MRNA for PCR amplification 00 000 cc cece eee eee eee ees 12 PG Ream plilication scx 2e ieaie a Beene se ee oe ee Sih oe ee ee Oe eee ae 13 mRNA isolation from tumor cells cece nent nn t eee arran 14 Before you begin asoeio rasaan a askew ne de et a sete eee Sema eed Cee 14 Prepare lysate from tumor cells isolated from whole blood or MNC 14 Isolate MRN
8. AMV resulting in greater enzyme specific activity than native AMV RT leading to higher cDNA yields and improved sensitivity 12328 019 ThermoScript Reverse Transcriptase An avian RT with reduced RNase H activity designed for RT PCR 12236 014 Dynabeads mRNA DIRECT Micro Kit User Guide 31 Appendix C Ordering Information Additional products mentioned in this user guide Additional products mentioned in this user guide Item Description Life Technologies Cat no lon Total RNA Seq Kit v2 Use to convert RNA transcripts expressed in a cell or tissue into representative cDNA libraries for strand specific RNA sequencing on the lon Torrent Personal Genome Machine PGM System 4475936 Exfold ERCC Spike In Mix ERCC RNA Spike In Mix Use these kits to e Measure sensitivity lower limit of detection and dynamic range of an experiment e Quantitate differential gene expression e Ensure quality control amongst experiments 4456739 4456740 Qubit RNA Assay Kit Provides an accurate and selective method for the quantitation of high abundance RNA samples Q32852 32 Dynabeads mRNA DIRECT Micro Kit User Guide Safety WARNING GENERAL SAFETY Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device Ensure that anyone using this product has received instructions i
9. C Note If you will use the isolated mRNA to perform RT PCR we recommend that you do not elute the mRNA instead use the Dynabeads Oligo dT 75 mRNA complex immediately for reverse transcription see PCR amplification on page 11 If storage is needed elute and store the mRNA as directed in Guidelines for best results on page 26 Next steps if using Do not quantify or qualify the isolated mRNA Proceed directly to the step Fragment lon Total RNA Seq the RNA using RNase III in the low input RNA Seq whole transcriptome library Kit v2 preparation procedure described in the Ion Total RNA Seq Kit v2 User Guide Part no 4476286 Use the entire eluted volume of mRNA as your starting material Note You can assume 1 recovery of original total RNA input Dynabeads mRNA DIRECT Micro Kit User Guide 25 JAN Guidelines and Troubleshooting Guidelines for best results e Prevent RNase contamination by following standard procedures during the preparation of starting material and during the experiment for example Wear disposable gloves and change them frequently Use sterile RNase free microcentrifuge tubes and pipette tips e Keep the vials of Dynabeads Oligo dT y5 in an upright position to ensure that the beads are covered with buffer as drying will reduce their performance Note If the Dynabeads Oligo dT 5 become dried out resuspend the beads in the buffer they are supplied in by placing the vial
10. Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support techsupport lifetech com e Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents e Obtain information about customer training e Download software updates and patches Limited Product Warranty Life Technologies Corporation and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support Dynabeads mRNA DIRECT Micro Kit User Guide 37 38 Bibliography Fellmann F et al 1996 Simplified Protocol of Solid Phase cDNA Libraries for Multiple PCR Amplification BioTechniques 21 766 770 Fiorenza M T and Mangia F 1998 Quantitative RT PCR Amplification of RNA in Single Mouse oocytes and preimplantation Embryos BioTechniques 24 618 623 Jakobsen K S et al 1994 Direct mRNA isolation using Magnetic Oligo dT Beads A protocol for all types of cell cultures animal and plant tissues In Advances in Biomagnetic Separation Editors Uhl n M Hornes E and Olsvik Natick MA Eaton Publishing 61 72 Karrer E E et al 1995 In S
11. by adding different specific primer sets in successive PCR reactions using the solid phase cDNA library as template Some of the advantages are e Allows multiple analysis of precious materials and small samples e One extraction allows the amplification of several gene transcripts e Enables simple and rapid buffer changes required to optimize the conditions for specific enzymes PCR amplification from a reusable solid phase cDNA library 1 Add PCR mix with primers and Taq polymerase and resuspend the cDNA Dynabeads properly Cycle twice to generate enough template for further amplification with a5 minute extension at 72 C Melt the strands at 94 C for 2 minutes place on magnet and transfer the supernatant with the amplification product to a new PCR tube Continue the cycling reaction 2 Wash the cCDNA Dynabeads twice in 10 mM Tris HCl or 1 x PCR buffer and reuse them by adding a new PCR mix for amplification of a different transcript 3 For storage of the cDNA library use TE buffer or equivalent amplified PCR products are analyzed by standard molecular methods Note If it is not necessary to reuse the CDNA Dynabeads just run the PCR with the beads present through the cycling reactions as described RNA Seg whole transcriptome library preparation Use one of the protocols in Chapter 3 mRNA Isolation from Purified Total RNA on page 16 to prepare mRNA samples from low input 100 ng 1 ug or standard input gt
12. heat denatured RNA mixture to each well that contains Dynabeads Oligo dT z5 Dynabeads mRNA DIRECT Micro Kit User Guide 19 3 Chapter 3 mRNA Isolation from Purified Total RNA mRNA isolation from gt 1 50 ug total RNA samples c Pipet the mixture up and down 10 times then incubate at room temperature for 5 minutes The mixture should appear homogeneous d Place the plate on the Magnetic Stand 96 After the solution clears remove and discard the supernatant without disturbing the pellet 3 Wash the RNA IMPORTANT LiDS a component of the Lysis Binding and Washing A Buffers is a strong inhibitor of enzymatic reactions To minimize LiDS carryover Always use Washing Buffer A first followed by Washing Buffer B Washing Buffer B does not contain LiDS Thoroughly resuspend the beads mRNA complex during the wash steps a Remove the plate from the Magnetic Stand 96 Add the appropriate amount of Washing Buffer A to each well then pipet up and down 10 times Total RNA input Washing Buffer A volume 1 8 ug 200 uL gt 8 50 ug 600 uL The mixture should appear homogeneous b Place the plate on the Magnetic Stand 96 After the solution clears remove and discard the supernatant without disturbing the pellet c Remove the plate from the Magnetic Stand 96 Add 300 uL of Washing Buffer B to each well then pipet up and down 10 times The mixture should appear homogeneous d Place the plate on the Magn
13. on a roller or equivalent overnight 2 C to 8 C This treatment will restore their complete functionality e RNase inhibitors may be added to the protocol at any step Note Adding an RNase inhibitor is normally redundant e If you are isolating mRNA for RT PCR We recommend that you immediately use the Dynabeads mRNA complex for reverse transcription If storage is needed elute the mRNA off the beads and freeze the mRNA containing supernatant When storing mRNA it is critical that no RNase is present in your sample If elution of mRNA is necessary add 10 20 uL 10 mM Tris HCl and incubate at 65 C to 80 C for 2 minutes Place the tube on the magnet and immediately transfer the supernatant to a new microcentrifuge tube The eluate may be used directly for reverse transcription or frozen for later use 80 C e If needed measure the concentration of mRNA by reading the absorbance of eluted mRNA at 260 nm The solution must be free of Dynabeads Oligo dT 95 as the beads will interfere with the spectrophotometrical readings e For mRNA isolation from larger sample volumes use the Dynabeads mRNA Direct Kit 26 Dynabeads mRNA DIRECT Micro Kit User Guide Troubleshooting Appendix A Guidelines and Troubleshooting Troubleshooting Observation Possible cause Recommended action Dynabeads Oligo dT 95 are dried out Dynabeads Oligo dT 5 were not capped correctly or were not stored in an upright po
14. which is not used in this protocol Confirm that the Lysis Binding Buffer has not precipitated If any precipitation is observed warm to room temperature and shake to dissolve Warm 50 uL nuclease free water per sample at 80 C Set a heat block or water bath to 70 C Wash the Note Keep the Dynabeads Oligo dT 95 in liquid suspension during handling and Dyna beads Oligo storage to avoid reduced performance dT o5 1 Vortex the Dynabeads Oligo dT 5 thoroughly to resuspend 2 Pipet the appropriate volume of beads 22 uL of beads per sample into a new 1 5 mL tube Note If you are processing a single sample you can pipet 22 uL of beads into a single well in a 1 2 mL 96 well plate Use separate wells for preparing the Dynabeads and for isolating the mRNA 3 Place the tube onto a magnetic stand then allow the suspension to clear 4 Aspirate and discard the clear supernatant without disturbing the beads 5 Remove the tube from the magnetic stand then add an equivalent volume of fresh Lysis Binding Buffer to the beads 6 Pipet up and down 10 times to resuspend the beads Avoid splashing or introducing bubbles to the suspension The mixture should appear homogeneous Prepa re the total Note Use the highest quality total RNA possible as your starting material Ideally use RNA sample s RNA with an RNA integrity number RIN greater than 7 Use FirstChoice Total RNA for high quality intact RNA isolated from a variety of sources
15. 000 Provides economical access to a range of high quality tissue specific total RNAs See www lifetechnologies com for a full list of available products PureLink RNA Mini Kit Column based method for isolating high quality total RNA from animal and plant cells and tissues as well as blood bacteria yeast and liquid samples 12183018A mirVana miRNA Isolation Kit Uses a rapid procedure to isolate small RNAs from tissue and cells using an efficient glass fiber filter GFF based method AM1560 MagMAX 96 for Microarrays Total RNA Isolation Kit Provides rapid high throughput purification of RNA from mammalian cells and tissue in 96 well plates AM1839 30 Dynabeads mRNA DIRECT Micro Kit User Guide Appendix C Ordering Information Reverse transcription for Real Time PCR products Reverse transcription for Real Time PCR products Item Description Life Technologies Cat no High Capacity cDNA Reverse Transcription Kit 1000 Reactions Formerly the High Capacity cDNA Archive Kit Contains all components necessary for the quantitative conversion of up to 2 ug of input total RNA in a single 20 uL reaction to single stranded cDNA Reactions can be scaled up to 100 uL to generate 10 ug of cDNA from a single reaction 4368813 High Capacity RNA to cDNA Kit High quality reverse transcription for easy reaction 4387406 set up and short reaction time with max
16. 1 50 ug total RNA The resulting sample is appropriate for use with the Ion Total RNA Seq Kit v2 0 Part no 4475936 which converts the RNA transcripts into representative cDNA libraries for strand specific RNA sequencing on the Ion Personal Genome Machine PGM sequencer Dynabeads mRNA DIRECT Micro Kit User Guide 29 Ordering Information Other Dynabeads Oligo dT 25 products 0 eee eee 30 Total RNA and total RNA purification products 0 eee eee ee 30 Reverse transcription for Real Time PCR products 0005000 31 Reverse transcription for cloning products 6 c cece eee eee 31 Additional products mentioned in this user guide 006 32 Other Dynabeads Oligo dT products Item Description Life Technologies Cat no Dynabeads mRNA DIRECT Kit For direct isolation of mRNA from cells animal and plant tissue 61011 and 61012 Dynabeads mRNA Purification Kit For mRNA purification from total RNA 61006 Dynabeads Epithelial Enrich For enriching epithelial tumor cells directly from 16102 whole blood bone marrow or PBMC suspensions Dynabeads Oligo dT 95 2x 1mL 61002 Dynabeads Oligo dT o5 5x 1mL 61005 Total RNA and total RNA purification products Item Description Life Technologies Cat no Ambion Total RNA products for example FirstChoice Human Total RNA Survey Panel AM6
17. 150 uL from table in step 2 gt 8 50 ug 300 uL volume Spike In Mix 300 uL from table in step 2 t Ifyou will add spike in control mix see next step reduce the final RNA sample volume to allow for the appropriate volume of spike in mix For example for 5 ug Total RNA input bring the sample volume to 149 uL to allow for the addition of 1 uL spike in mix in the next step 2 Recommended for gene expression experiments Add an ERCC Spike In Control Mix to the diluted total RNA sample See the ERCC RNA Spike In Control Mixes User Guide Part no 4455352 for mix selection and sample preparation procedures The following chart indicates the correct dilution of ERCC to be added to the total RNA sample based on input Total RNA input Volume of Spike In Mix 1 or Mix 2 dilution 1 ug 2 uL 1 100 5 ug 1 uL 1 10 10 ug 1 uL 1 5 20 ug 2 uL 1 5 30 ug 3 pL 1 5 40 ug 4uL 1 5 50 yg Tul t ERCC RNA Spike In Mix 1 ExFold Spike In Mix 1 or ExFold Spike In Mix 2 Perform two 1 Prepare the RNA for binding rounds of mRNA a Heat the total RNA at 70 C for 2 minutes isolation in the b Add an equal volume of Lysis Binding Buffer to each volume of prepared same well total RNA sample mix well then quick spin the samples 2 Bind the mRNA to the Dynabeads a Pipet the appropriate volume of washed Dynabeads Oligo dT 95 into a new well of the 1 2 mL 96 well plate b Transfer the
18. 50 Tubes 1 5 mL 250 Heat block and or incubator MLS mRNA isolation from gt 1 50 ug total RNA samples Before you begin e Read Guidelines for best results on page 26 e Prepare the Ion Total RNA Seq Kit v2 reagents as directed in the Jon Total RNA Seq Kit v2 User Guide Pub no 4476286 before beginning this mRNA isolation protocol e Bring all the Dynabeads mRNA DIRECT Micro Kit reagents to room temperature except the 10 mM Tris HCl which is not used in this protocol e Confirm that the Lysis Binding Buffer has not precipitated If any precipitation is observed warm to room temperature and shake to dissolve e Warm 180 uL nuclease free water per sample to 80 C e Set a heat block or water bath to 70 C Dynabeads mRNA DIRECT Micro Kit User Guide 17 3 Chapter 3 mRNA Isolation from Purified Total RNA mRNA isolation from gt 1 50 ug total RNA samples Wash the Note Keep the Dynabeads Oligo dT y5 in liquid suspension during handling and Dyna beads Oligo storage to avoid reduced performance dT o5 1 18 Vortex the Dynabeads Oligo dT 95 thoroughly to resuspend 2 Pipet the appropriate volume of beads for your total RNA input into a new 1 5 mL tube or 1 2 mL 96 well U bottom plate Note Include 5 10 excess volume for pipetting losses For example for 6 samples of 5 ug total RNA input add 6 x 30 uL x 1 05 190 uL of beads to the tube Volume per sample
19. A for PCR amplification 00 00000 cece eens 14 PGR am pluinicatlon a Scr n Ain oh 23 meth Mee itis la a RED aaah oe een tN 2d 28 ed coat mare 15 CHAPTER 3 mRNA Isolation from Purified Total RNA 16 WoOPkKHOW 3 nce esa wc Seer oe Sede ea wee eo Side ete a eaten 16 Materials and equipment required but not provided 00 eect ee ees 17 mRNA isolation from gt 1 50 pg total RNA samples 00000 c cece cece eee es 17 Before you begin usarem geen os Ma eae eon eet bowie alec ls Sei 17 Wash the Dynabeads Oligo dT o5 2 cece nen eee eee eee aes 18 Dynabeads mRNA DIRECT Micro Kit User Guide 3 Contents Prepare the total RNA samplels 000 c ccc cece cnc nen rnrn nen eens 19 Perform two rounds of mRNA isolation in the same well 000 e cece eee eens 19 Assess the yield and size distribution of the MRNA 000000 cece cere eens 22 mRNA isolation from 100 ng 1 pg total RNA samples 000 ccc cece 23 Before Vou begun ian EE tint 6 seh eteca ied ded bee ak eh E Bere Gy cid are mene diate catia 23 Wash the Dynabeads Oligo dT os 2 0 0 cece eect ee eee e eee e teen teen teen tees 23 Prepare the total RNA samplels 00000 cece cece cence cence enn e eee n eee 23 Perform two rounds of mRNA isolation in the same well 00 e cence eee eens 24 Next steps if using lon Total RNA Seq Kit v2 00 020 e eee eee eee eee
20. Guide 35 Documentation and Support Related documentation Obtaining SDSs The following related document is available from http ioncommunity iontorrent com Document Ratt Description number lon Total RNA Seq Kit v2 4476286 Provides detailed procedures for RNA Seq whole User Guide transcriptome library preparation using the lon Total RNA Seq Kit v2 Note To open the user documentation available from http ioncommunity iontorrent com use the Adobe Reader software available from www adobe com Note For additional documentation see Obtaining support on page 37 Safety Data Sheets SDSs are available from www lifetechnologies com support Note For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufacturer Obtaining Certificates of Analysis 36 The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www lifetechnologies com support and search for the Certificate of Analysis by product lot number which is printed on the box Dynabeads mRNA DIRECT Micro Kit User Guide Documentation and Support Obtaining support Obtaining support For the latest services and support information for all locations go to www lifetechnologies com support At the website you can e Access worldwide telephone and fax numbers to contact Technical
21. Micro Kit User Guide 5 Chapter 1 Product Information Contents and storage Product The Dynabeads mRNA DIRECT Micro Kit provides enough reagents for performance e 100 microscale direct mRNA isolations from up to 2 5 x 104 mononuclear cells up to 1 x 104 cultured cells or up to 5 mg tissue depending on the tissue type e 100 microscale or 20 large scale 50 ug mRNA isolations from purified total RNA A strong RNase inhibiting agent and stringent hybridization and washing buffers ensure isolation of intact high purity mRNA even from crude samples rich in RNases Enzymatic downstream applications are not inhibited by the presence of the beads 1 mL of Dynabeads Oligo dT y5 can isolate 10 ug of mRNA The specific yield will depend on the tissue cell or total RNA type Contents and storage The Dynabeads mRNA DIRECT Micro Kit Cat no 61021 contains the following components Contents Description Storage Conditions Dynabeads Oligo dT o5 Supplied as approximately 5 mg mL bead suspension in Store at 2 C to 8 C After 2x 1mL phosphate buffered saline PBS pH 7 4 containing opening avoid bacterial 0 02 sodium azide NaN3 as a preservative contamination e 137 mM NaCl e 2 7 mM KCL e 43 mM NaHPO x 7H20 e 1 4 mM KH PO e 0 02 NaN3 Lysis Binding Buffer 15 mL e 100 mM Tris HCl pH 7 5 e 500 mM LiCl e 10 mM EDTA pH 8 0 e 1 LIDS e 5 mM dithiothreitol DTT Washing Buffer A 30 mL e 10 mM
22. NA at 70 C for 2 minutes isolation in the b Add 50 uL of Lysis Binding Buffer to each 50 uL prepared total RNA same well sample mix well then quick spin the samples 2 Bind the mRNA to the Dynabeads a Pipet 20 uL of washed Dynabeads Oligo dT 25 into a new well of the 1 2 mL 96 well plate b Transfer 100 uL of the heat denatured RNA mixture to the well that contains Dynabeads Oligo dT z5 c Pipet the mixture up and down 10 times then incubate at room temperature for 5 minutes The mixture should appear homogeneous d Place the plate on the Magnetic Stand 96 After the solution clears remove and discard the supernatant without disturbing the pellet 3 Wash the RNA IMPORTANT LiDS a component of the Lysis Binding and Washing A Buffers is a strong inhibitor of enzymatic reactions To minimize LiDS carryover Always use Washing Buffer A first followed by Washing Buffer B Washing Buffer B does not contain LiDS Thoroughly resuspend the beads mRNA complex during the wash steps a Remove the plate from the Magnetic Stand 96 Add 100 uL Washing Buffer A to each well then pipet up and down 10 times The mixture should appear homogeneous b Place the plate on the Magnetic Stand 96 After the solution clears remove and discard the supernatant without disturbing the pellet c Remove the plate from the Magnetic Stand 96 Add 100 uL Washing Buffer B to each well then pipet up and down 10 times The mixture
23. NA isolation but not less than 1 mL to the microcentrifuge tube then mix 2 Chapter 2 Direct mRNA Isolation from Microscale Samples from Cells or Tissues mRNA isolation from cultured cells and cell suspensions Prepare samples Place the tube on a magnet for 1 minute then remove and discard the supernatant Remove the tube from the magnet then resuspend the beads in the same volume of Lysis Binding Buffer that you used in step 2c 20 uL per mRNA isolation Aliquot 20 uL suspension to each sample tube If you are preparing reverse transcription reactions Prepare your reverse transcription PCR mix before the mRNA isolation and keep on ice If you are working with cells previously isolated using magnetic separation Make sure that all Dynabeads are removed from the lysate before adding Dynabeads Oligo dT zs5 see mRNA isolation from tumor cells on page 14 If you are working with frozen cells Perform a rapid lysis in Lysis Binding buffer This is critical for obtaining undegraded mRNA Avoid thawing of frozen material before lysis mRNA isolation from cultured cells and cell suspensions Before you begin Prepare lysate from cultured cells and cell suspensions Isolate MRNA for PCR amplification 10 IMPORTANT To prevent RNA degradation by RNase contamination use gloves and change them frequently Note To isolate mRNA from primary tumor cells from whole blood or bone marrow tissues see mRNA isolat
24. Tris HCl pH 7 5 e 0 15 M LiCl e 1 mM EDTA e 0 1 LIDS Washing Buffer B 30 mL e 10 mM Tris HCl pH 7 5 e 0 15 M LiCl e 1mM EDTA 10 mM Tris HClt 15 mL 10 mM Tris HCl pH 7 5 The protocols in Chapter 3 mRNA Isolation from Purified Total RNA on page 16 use nuclease free water for elution rather than the 10 mM Tris HCl Elution Buffer provided with the kit 6 Dynabeads mRNA DIRECT Micro Kit User Guide Storage and stability Dynabeads mRNA DIRECT Micro Kit User Guide Chapter 1 Product Information 1 Contents and storage The components in the kit are guaranteed stable until the expiry date stated on the label when stored unopened at 2 C to 8 C All reagents used are of analytical grade and RNase free The Dynabeads suspension and the buffers provided in the kit are ribonuclease free and tested for optimal performance Dynabeads Oligo dT y5 are stable in a pH range of 4 13 Dynabeads Oligo dT 25 may be frozen in the buffer they are supplied in Avoid repeated freezing and thawing Do not store or freeze the Dynabeads in distilled water Store the vials of Dynabeads upright to avoid drying of the beads If the Dynabeads have dried in the vial resuspend the beads in the buffer they are supplied in by placing the vial on a mixer overnight 4 C If the buffers precipitate warm to room temperature and mix until the precipitate dissolves Direct MRNA Isolation from Mic
25. USER GUIDE ambion oy life technologies Dynabeads mRNA DIRECT Micro Kit Catalog Number 61021 Revision 004 Revision Date 14 May 2012 o For Research Use Only Not for human or animal therapeutic or diagnostic use technologies For Research Use Only Not for human or animal therapeutic or diagnostic use The information in this guide is subject to change without notice DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF NOTICE TO PURCHASER LIMITED USE LABEL LICENSE Research Use Only The purchase of this product conveys to the purchaser the limited non transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser No right to resell this product or any of its components is conveyed expressly by implication or by estoppel This product is for internal research purposes only and is not for use in commercial a
26. aining 20 uL pre washed Dynabeads Oligo dT 95 prepared according to Wash Dynabeads Oligo dT 25 before use on page 9 3 Pipet up and down 2 3 times to mix 4 Place the tube on a sample mixer or roller for 5 minutes at room temperature to allow the mRNA to anneal to the Dynabeads with continuous rotation 5 Place the sample tube on the magnet for 2 minutes then discard the supernatant 6 Remove the sample tube from the magnet and resuspend the Dynabeads mRNA complex in 100 uL Washing Buffer A by careful pipetting 7 Place the sample tube on the magnet for 1 minute then discard the supernatant 8 Repeat steps 6 7 once 9 Resuspend the Dynabeads mRNA complex in 100 uL Washing Buffer B Dynabeads mRNA DIRECT Micro Kit User Guide 10 11 12 13 14 15 PCR amplification 1 Chapter 2 Direct mRNA Isolation from Microscale Samples from Cells or Tissues 2 mRNA isolation from tumor cells Transfer the suspension to a new tube Place the new sample tube on the magnet for 1 minute then discard the supernatant Resuspend the Dynabeads mRNA complex in 100 uL Washing Buffer B Place the sample tube on the magnet for 1 minute then discard the supernatant Remove the sample tube from the magnet and resuspend the Dynabeads mRNA complex in 100 uL of ice cold 10 mM Tris HCl Store the sample tube on ice for immediate use in PCR amplification Note We recommend that you immediately use the Dynab
27. e kit to isolate mRNA directly from microscale samples from cells or tissues isolation is completed in only 15 minutes in a single tube without the need to prepare total RNA or to perform any other purifications steps Use the isolated mRNA directly for reverse transcription into cDNA followed by transcript amplification by PCR The bead bound oligo dT is used both to capture the mRNA and as a primer for reverse transcriptase synthesis into first strand cDNA The isolated mRNA does not need to be eluted from the beads but can instead be used directly for reverse transcription and PCR amplification The combination of direct mRNA isolation and one tube reverse transcription allows for fast and reliable PCR detection When using the kit to isolate mRNA from purified total RNA two rounds of mRNA isolation are completed in less than 30 minutes The resulting isolated mRNA contains very low ribosomal RNA content which allows for the most accurate measurement of coding transcripts Samples are prepared in a plate to enable parallel processing of multiple samples The resulting isolated mRNA is ready for any downstream application and has been validated for RNA Seq whole transcriptome library preparation using the Ion Total RNA Segq Kit v2 Note All protocols in this user guide can be used to obtain a final product of eluted mRNA or a Dynabeads mRNA complex depending on the requirements for the downstream application Dynabeads mRNA DIRECT
28. e lysate 1 from plant and animal tissues Isolate mRNA for 1 PCR amplification pi 12 O o o ek SS Grind frozen tissue sample up to 5 mg depending on the tissue type in a microcentrifuge tube using a manual tissue grinder Work quickly Note Aliquot weigh the animal or plant tissue while frozen to avoid mRNA degradation Use the specified amount of tissue since an excess of tissue will reduce the mRNA yield and purity Keep the sample frozen by dipping the sample tube in liquid nitrogen Add 100 uL Lysis Binding Buffer and thaw sample while continuing to grind until complete lysis is obtained approximately 1 2 minutes A rapid lysis in the Lysis Binding Buffer is critical to obtain undegraded mRNA If the raw extract is noticeably viscous a shearing step might be beneficial see Troubleshooting on page 27 Centrifuge the lysate for 30 60 seconds in a microcentrifuge to remove debris The lysate can be frozen and stored at 80 C for later use Combine the 100 uL lysate with the 20 uL prewashed Dynabeads Oligo dT 25 prepared according to Wash Dynabeads Oligo dT 25 before use on page 9 Pipet up and down 2 3 times to mix Place the tube on a sample mixer or roller for 5 minutes at room temperature to allow the mRNA to anneal to the Dynabeads with continuous rotation Place the sample tube on the magnet for 1 minute then discard the supernatant Remove the sample tube from the magnet and r
29. e transcription PCR mix place the tube containing the Dynabeads mRNA complex on the magnet for 1 minute then discard the supernatant Add reverse transcription PCR reaction mix e For one tube PCR Resuspend the Dynabeads mRNA complex in 50 uL reverse transcription PCR mix and transfer to the PCR tube e For two step PCR Resuspend the Dynabeads mRNA complex in the reverse transcription PCR reaction mix according to the manufacturer s recommendation Perform cDNA synthesis as recommended by the manufacturer of the reverse transcriptase When using a thermostable reverse transcriptase and the bead bound oligo dT as primer for first strand cDNA synthesis an initial incubation at 50 C for 5 minutes is necessary before proceeding at the recommended temperature For additional guidelines on using the isolated mRNA in downstream applications see Appendix B on page 28 Dynabeads mRNA DIRECT Micro Kit User Guide 11 Chapter 2 Direct mRNA Isolation from Microscale Samples from Cells or Tissues mRNA isolation from tissues MRNA isolation from tissues IMPORTANT To prevent RNA degradation by RNase contamination use gloves and change them frequently Before you begin e Follow the instructions in Prepare reagents and samples on page 9 e Read Guidelines for best results on page 26 Note We recommend that you prepare the reverse transcription PCR mix before starting the mRNA isolation protocol Prepar
30. eads mRNA complex for reverse transcription If storage is needed elute the mRNA off the beads and freeze the mRNA containing supernatant When storing mRNA it is critical that no RNase is present in your sample If elution of mRNA is necessary add 10 20 uL 10 mM Tris HCl and incubate at 65 C to 80 C for 2 minutes Place the tube on the magnet and immediately transfer the supernatant to a new microcentrifuge tube The eluate may be used immediately for reverse transcription or store for up to two weeks at 80 C Immediately before adding the reverse transcription PCR mix place the tube containing the Dynabeads mRNA complex on the magnet for 1 minute then discard the supernatant Add reverse transcription PCR reaction mix e For one tube PCR Resuspend the Dynabeads mRNA complex in 50 uL reverse transcription PCR mix and transfer to the PCR tube e For two step PCR Resuspend the Dynabeads mRNA complex in the reverse transcription PCR reaction mix according to the manufacturer s recommendation Perform cDNA synthesis as recommended by the manufacturer of the reverse transcriptase When using a thermostable reverse transcriptase and the bead bound oligo dT as primer for first strand cDNA synthesis an initial incubation at 50 C for 5 minutes is necessary before proceeding at the recommended temperature For additional guidelines on using the isolated mRNA in downstream applications see Appendix B on page 28 Dynabeads
31. eee 25 APPENDIX A Guidelines and Troubleshooting 26 Guidelines for best results 0 0 cece cece ene tenet eee ERRA ees 26 Troubleshooting asien hd back bye ree E a ad Later da ted hn aad ena ie cote 27 APPENDIX B Downstream Applications 000ce eens 28 Solid phase cDNA synthesis and one tube reverse transcription PCR 2 20 28 Construction of immobilized cDNA libraries for multiple PCR amplifications 29 PCR amplification from a reusable solid phase cDNA library 0 00000e cee eee eres 29 RNA Seq whole transcriptome library preparation 0000 ccc cece ee eee eee ees 29 APPENDIX Ordering Information 0ce cece eee eeee 30 Other Dynabeads Oligo dT o5 products 20 c cece cece tence ene rrn 30 Total RNA and total RNA purification products 0 0 0 0 cece cece eects 30 Reverse transcription for Real Time PCR products 00 0000 eee eee eee e eee aee 31 Reverse transcription for cloning products 2 20000 cece cece eee eee eee e eens 31 Additional products mentioned in this user guide 0 000cc eee eee eee ees 32 APPENDIX D Safety wiscccxvcaii estates ty eewt ed reena 33 Chemicalsafety lt nencai a iaa a an eed nabhade bs Seb a pi Sod ea a eek hc a E 34 Specific chemical handling eenei sees ond Seagate de eared eed be eee 34 Biological hazard safety 020 c cece eee e eee e nent e ee
32. esuspend the Dynabeads mRNA complex in 100 uL Washing Buffer A by careful pipetting Place the sample tube on the magnet for 1 minute then discard the supernatant Repeat steps 5 6 once Resuspend the Dynabeads mRNA complex in 100 uL Washing Buffer B Transfer the suspension to a new tube Place the new sample tube on the magnet for 1 minute then discard the supernatant Resuspend the Dynabeads mRNA complex in 100 uL Washing Buffer B 12 Place the sample tube on the magnet for 1 minute then discard the supernatant Dynabeads mRNA DIRECT Micro Kit User Guide Chapter 2 Direct mRNA Isolation from Microscale Samples from Cells or Tissues 2 mRNA isolation from tissues 13 Remove the sample tube from the magnet and resuspend the Dynabeads mRNA complex in 100 uL of ice cold 10 mM Tris HCl 14 Store the sample tube on ice for immediate use in PCR amplification Note We recommend that you immediately use the Dynabeads mRNA complex for reverse transcription If storage is needed elute the mRNA off the beads and freeze the mRNA containing supernatant When storing mRNA it is critical that no RNase is present in your sample If elution of mRNA is necessary add 10 20 uL 10 mM Tris HCl and incubate at 65 C to 80 C for 2 minutes Place the tube on the magnet and immediately transfer the supernatant to a new microcentrifuge tube The eluate may be used immediately for reverse transcription or store for up to two weeks at
33. etic Stand 96 After the solution clears remove and discard the supernatant without disturbing the pellet IMPORTANT Remove as much Washing Buffer B as possible without disturbing the pellet before you elute the samples 4 Elute the mRNA a Remove the plate from Magnetic Stand 96 Add the specified volume of warmed 80 C nuclease free water to each well pipet up and down 10 times to resuspend the beads in the nuclease free water then let sit 30 seconds at room temperature Volume warmed nuclease free Tekan ecb water to add per sample 1 20 ug 50 uL gt 20 50 ug 90 uL 20 Dynabeads mRNA DIRECT Micro Kit User Guide Chapter 3 mRNA Isolation from Purified Total RNA 3 mRNA isolation from gt 1 50 ug total RNA samples 5 Re bind the mRNA to the Dynabeads a Add an equal volume of Lysis Binding Buffer to each well then pipet up and down 10 times b Incubate at room temperature for 5 minutes The mixture should appear homogeneous c Place the plate on the Magnetic Stand 96 After the solution clears remove and discard the supernatant without disturbing the pellet 6 Repeat step 3 to wash the RNA 7 Elute the mRNA a Remove the plate from the Magnetic Stand 96 then add the specified volume of the pre heated 80 C nuclease free water to each well Volume warmed nuclease free Total RNA input water to add per sample 1 8 ug 10 uL gt 8 20 ug 10 15 uL gt 20 50 ug 20 30 uL
34. imum sensitivity and dynamic range Platinum Quantitative RT PCR Real time quantitation of RNA molecules from total 11731 015 ThermoScript One Step System or poly A RNA in a single step SuperScript VILO cDNA Variable input linear output to increase the dynamic 11754 050 Synthesis Kit range of qRT PCR assays SuperScript VILO Master Mix Reverse transcriptase premix containing everything 11755050 needed for reverse transcription RT in one tube Reverse transcription for cloning products Item Description Life Technologies Cat no M MLV Reverse Transcriptase M MLV Moloney Murine Leukemia Virus Reverse Transcriptase RT is a recombinant DNA polymerase that synthesizes a complementary DNA strand from single stranded RNA DNA or an RNA DNA hybrid 28025 013 SuperScript II Reverse Transcriptase A DNA polymerase that synthesizes a complementary DNA strand from single stranded RNA DNA or an RNA DNA hybrid 18064 014 SuperScript III Reverse Transcriptase A proprietary mutant of SuperScript II RT that is active at 50 C and has a half life of 220 minutes providing increased specificity with Gene Specific Primers GSPs and the highest cDNA yield of all RTs 18080 044 AMV Reverse Transcriptase Cloned Cloned Avian Myeloblastosis Virus AMV Reverse Transcriptase RT is highly purified from insect cells infected with baculovirus containing the pol gene of
35. ing materials are also required Liquid nitrogen MLS Manual tissue grinder MLS Syringe and needle see Troubleshooting on page 27 MLS When working with tumor cell samples the following is also required Dynabeads Epithelial Enrich Life Technologies 16102 Prepare reagents and samples IMPORTANT We strongly recommend that you read this section and Guidelines for best results on page 26 before starting your mRNA isolation protocol Prepare buffers Wash Dynabeads Oligo dT 25 before use Dynabeads mRNA DIRECT Micro Kit User Guide Bring all buffers except the 10 mM Tris HCl to room temperature before use Store the 10 mM Tris HCI on ice or at 2 C to 8 C before use Confirm that the Lysis Binding Buffer has not precipitated If any precipitation is observed warm to room temperature and shake to dissolve Resuspend the Dynabeads Oligo dT 95 in the tube either vortex for gt 30 seconds or tilt and rotate for 5 minutes Note If the Dynabeads Oligo dT 5 becomes dried out resuspend the beads in the buffer they are supplied in by placing the vial on a roller or equivalent overnight 2 C to 8 C to restore their complete functionality Transfer the appropriate volume of Dynabeads Oligo dT 5 use 20 uL Dynabeads per mRNA isolation from the stock tube suspension to an RNase free microcentrifuge tube Add an equivalent volume of Lysis Binding Buffer 20 uL per mR
36. ion from tumor cells on page 14 e Follow the instructions in Prepare reagents and samples on page 9 e Read Guidelines for best results on page 26 Note We recommend that you prepare the reverse transcription PCR mix before starting the mRNA isolation protocol This protocol is recommended for samples containing up to 1 x 104 cultured cells or up to 2 5 x 104 mononuclear cells 1 Wash the cell suspension in phosphate buffered saline PBS prior to preparing a cell pellet by centrifugation The cell pellet can be used immediately or frozen in liquid nitrogen and stored at 80 C for later use Add 100 uL Lysis Binding Buffer to the fresh frozen cell pellet Perform a repeated passage of the solution through a pipette tip to obtain complete lysis The lysate may be frozen 80 C and stored for later use Transfer the clear lysate to the tube containing 20 uL pre washed Dynabeads Oligo dT 25 prepared according to Wash Dynabeads Oligo dT 25 before use on page 9 Pipet up and down 2 3 times to mix Place the tube on a sample mixer or roller for 5 minutes at room temperature to allow the mRNA to anneal to the Dynabeads with continuous rotation Dynabeads mRNA DIRECT Micro Kit User Guide en 11 12 13 14 PCR amplification 1 o 30 oN Chapter 2 Direct mRNA Isolation from Microscale Samples from Cells or Tissues 2 mRNA isolation from cultured cells and cell suspensions
37. itu isolation of mRNA from individual plant cells Creation of cell specific cDNA libraries Proc Natl Acad Sci USA 92 3814 3818 Lambert K N and Williamson V M 1993 cDNA library construction from small amounts of RNA using paramagnetic beads and PCR Nucleic Acids Res 21 775 776 Lee Y H and Vacquier V D 1992 Reusable cDNA libraries coupled to magnetic beads Anal Biochem 206 206 207 Raineri I et al 1991 Improved efficiency for single sided PCR by creating a reusable pool of first strand cDNA coupled to a solid phase Nucleic Acids Research 19 4010 Raineri I and Senn H P 1992 HIV 1 promotor insertion revealed by selective detection of chimeric provirus host gene transcripts Nucleic Acids Res 20 6261 6266 Sharma et al 1993 PCR based construction of subtractive cDNA library using magnetic beads BioTechniques 15 610 611 Dynabeads mRNA DIRECT Micro Kit User Guide Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit www lifetechnologies com support or email techsupport dlifetech com technologies www lifetechnologies com SPEC 06241 14 May 2012
38. le local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following In the U S U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety WHO_CDS_CSR_LYO_2004_11 en Dynabeads mRNA DIRECT Micro Kit User
39. most cDNA synthesis kits commercially available Perform cDNA synthesis as recommended by the manufacturer of the reverse transcriptase When using thermostable reverse transcriptase and the bead bound oligo dT as primer for first strand cDNA synthesis an initial step of incubation at 50 C for 5 minutes is necessary before proceeding at recommended temperature The PCR is not inhibited by the presence of the Dynabeads cDNA synthesis and PCR can be performed sequentially in one tube that is in the same reaction buffer using an enzyme capable of both RNA and DNA dependant polymerization The combination of direct mRNA isolation using Dynabeads Oligo dT 5 and one tube reverse transcription PCR offers a convenient system for fast and reliable PCR detection 28 Dynabeads mRNA DIRECT Micro Kit User Guide Appendix B Downstream Applications B Construction of immobilized cDNA libraries for multiple PCR amplifications Construction of immobilized cDNA libraries for multiple PCR amplifications A reusable solid phase cDNA library can be made directly on the bead surface The first strand cDNA using the bead bound oligo dT as primer is covalently linked to the Dynabeads Jakobsen et al 1994 Raineri et al 1991 Raineri et al 1992 Sharma et al 1993 Fellman et al 1996 and Karrer et al 1995 and can be reused for multiple PCR amplifications of specific transcripts The different transcripts are amplified
40. n page 11 If isolating mRNA from total RNA remove inhibitors by repeating washing steps starting with step 5 Re bind the mRNA to the Dynabeads on page 21 for gt 1 50 pg total RNA samples or step 5 Re bind the mRNA to the Dynabeads on page 25 for 100 ng 1 ug total RNA samples Dynabeads mRNA DIRECT Micro Kit User Guide 27 Downstream Applications Solid phase cDNA synthesis and one tube reverse transcription PCR 28 Construction of immobilized cDNA libraries for multiple PCR amplifications 29 PCR amplification from a reusable solid phase cDNA library 29 RNA Seq whole transcriptome library preparation 0 00 00 eens 29 Solid phase cDNA synthesis and one tube reverse transcription PCR Enzymatic downstream applications are not inhibited by the presence of Dynabeads Oligo dT 5 hence the bead bound mRNA can be used directly for solid phase cDNA synthesis and reverse transcription PCR Jakobsen et al 1994 Lambert et al 1993 Lee et al 1992 Raineri et al 1991 Raineri et al 1992 and Sharma et al 1993 The oligo dT sequence on the Dynabeads is used not only to capture the mRNA but also as a primer for the subsequent reverse transcriptase synthesis into CDNA The resulting first strand cDNA is covalently linked to the surface of the Dynabeads and may be used for cDNA amplification The Dynabeads solid phase technology is compatible with
41. n general safety practices for laboratories and the safety information provided in this document Before using an instrument or device read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection etc To obtain SDSs see the Documentation and Support section in this document Dynabeads mRNA DIRECT Micro Kit User Guide 33 Appendix D Safety Chemical safety Chemical safety WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials To obtain SDSs see the Documentation and Support section in this document Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Check reg
42. nal antibody BerEP4 against the human epithelial antigen HEA enriches epithelial tumor cells from blood or MNC 5 mL anticoagulated whole blood samples or mononuclear cells MNC at a concentration of 1 2 x 107 cells mL are mixed with the Dynabeads Epithelial Enrich The cells are isolated and purified by using a magnet and the cells can be directly lysed while still on the beads Isolate mRNA as described in the following procedure The isolated mRNA can also be used in PCR for other genes e Follow the instructions in Prepare reagents and samples on page 9 e Read Guidelines for best results on page 26 Note We recommend that you prepare the reverse transcription PCR mix before starting the mRNA isolation protocol 1 Follow the protocol for positive isolation of tumor cells from whole blood or MNC using Dynabeads Epithelial Enrich 2 After adding the final washing solution transfer the bead suspension to a microcentrifuge tube Keep the Dynabeads cell complex on ice for immediate mRNA isolation and PCR 3 Immediately before use place the sample tube on the magnet for 2 3 minutes and remove supernatant 4 Lyse the cells by resuspending the Dynabeads cell complex in 100 uL Lysis Binding Buffer 5 Pipet up and down 2 3 times to mix 1 Place the sample tube with the lysed cells on the magnet for 2 minutes 2 Transfer the supernatant clear lysate containing released mRNA to a microcentrifuge tube cont
43. pplications of any kind including without limitation quality control and commercial services such as reporting the results of purchaser s activities for a fee or other form of consideration For information on obtaining additional rights please contact outlicensingfalifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 Manufactured by Life Technologies AS Norway Life Technologies AS complies with the Quality System Standards ISO 9001 2008 and ISO 13485 2003 TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Agilent is a registered trademark and Bioanalyzer is a trademark of Agilent Technologies Inc Adobe and Adobe Reader are registered trademarks of Adobe Systems Inc 2012 Life Technologies Corporation All rights reserved Contents M CHAPTER 1 Product Information 00c eee cece eee 5 Purpose of products cena sic ghee aeek t een Laue edie eagles RE dase 5 Product performance ni reketo dese wages debe bet e be Soe ead ave ea a Ta Ride eee 6 Contents and storage 2 tesa stared bint E ey bee ee eh eee eee cd bets 6 Storage and stability 0 0 cece eee t teenies 7 CHAPTER 2 Direct mRNA Isolation from Microscale Samples from Cells or Tissuessc inu care eweeie nc une tas wa tenum ante 8 WOPK TOW 3 suo feces 3h a cates 3 Ai ce rg A Ae cena Ae na tN oa ale is Pon ade ce 2 Adee se cand at alg Be
44. roscale Samples from Cells or Tissues m WOT TOW e abide tacit ee Soe ET E ottah asec tong ile arent eds pene O E 8 m Materials and equipment required but not provided 00005 9 m Prepare reagents and samples 0066 e ccc 9 E mRNA isolation from cultured cells and cell suspensions 5 10 E mRNA isolation from tissues 0 eens 12 E mRNA isolation from tumor cells 0 cee 14 Workflow Prepare reagents and samples Prepare lysate Isolate mRNA for PCR amplification Prepare RNA for binding Bind mRNA to the Dynabeads Wash the mRNA Resuspend mRNA and store on ice for downstream applications Place mRNA on magnet and remove supernatant 8 Dynabeads mRNA DIRECT Micro Kit User Guide Chapter 2 Direct mRNA Isolation from Microscale Samples from Cells or Tissues 2 Materials and equipment required but not provided Materials and equipment required but not provided All reagents used should be analytical grade and RNase free Item Source Cat no DynaMag 2 Magnet or other appropriate magnet Life Technologies 12321D See www lifetechnologies com magnets for magnet recommendations Pipettors and sterile RNAse free pipette tips Major laboratory suppliers MLS Sterile RNase free microcentrifuge tubes MLS HulaMixer Sample Mixer Life Technologies 15920D When working with tissue samples the follow
45. s 35 Documentation and Support cece eee ee ees 36 Related documentation 2 2c004 tuenda enced wend Abe PES ee ee des ee cena eee 36 ObtalnINnG SDSShfa sor ora tid E A ied 5 clan Paes of td aah tak ES ca bec ned Bel sta od ike 36 Obtaining Certificates of Analysis 0000 c ccc eee aee 36 Obtaining Support csie oe Sasies vad decline gene ee ee baa eae dees Le pete tele 37 Limited Product Warranty 2 20 0c eee eee eee eee e nents 37 Bibliography ceesre nimni emt heen teens eee eee beams 38 Dynabeads mRNA DIRECT Micro Kit User Guide Product Information IMPORTANT Before using this product read and understand the information in Appendix D Safety on page 33 Purpose of product The Dynabeads mRNA DIRECT Micro Kit is designed for simple and rapid isolation of pure intact polyadenylated poly A mRNA The kit isolates highly purified and intact mRNA directly from microscale samples from cells or tissues and from purified total RNA samples The kit contains Dynabeads Oligo dT 95 uniform and superparamagnetic beads with oligo dT sequences covalently bound to their surface The isolation protocol relies on base pairing between the poly A residues at the 3 end of most mRNA and the oligo dT 5 residues covalently coupled to the surface of the Dynabeads Other RNA species lacking a poly A tail do not hybridize to the beads and are readily washed away When using th
46. should appear homogeneous 24 Dynabeads mRNA DIRECT Micro Kit User Guide Chapter 3 mRNA Isolation from Purified Total RNA 3 mRNA isolation from 100 ng 1 ug total RNA samples d Place the plate on the Magnetic Stand 96 After the solution clears remove and discard the supernatant without disturbing the pellet IMPORTANT Remove as much Washing Buffer B as possible without disturbing the pellet before you elute the samples 4 Elute the mRNA a Remove the plate from Magnetic Stand 96 b Add 25 uL of the pre heated 80 C nuclease free water to each well pipet 10 times to resuspend the beads in the nuclease free water then let sit 30 seconds at room temperature 5 Re bind the mRNA to the Dynabeads a Add 25 uL of Lysis Binding Buffer to each well then pipet up and down 10 times b Incubate at room temperature for 5 minutes The mixture should appear homogeneous c Place the plate on the Magnetic Stand 96 After the solution clears remove and discard the supernatant without disturbing the pellet 6 Repeat step 3 to wash the RNA 7 Elute the mRNA a Remove the plate from the Magnetic Stand 96 then add 10 uL of the warmed 80 C nuclease free water to each well b Place the plate on the Magnetic Stand 96 After the solution clears transfer the supernatant containing the mRNA to a new tube without disturbing the pellet Store the isolated mRNA on ice for immediate use or store for up to two weeks at 80
47. sition Resuspend the beads in the buffer they are supplied in by placing the vial on a roller or equivalent overnight 2 C to 8 C This treatment will restore their complete functionality Lysate is noticeably VISCOUS Released DNA or DNA contamination Note Direct mRNA isolation methods have a potential risk of DNA contamination The Dynabeads mRNA DIRECT Micro Kit protocol used only small amounts of cells and tissue and consequently this potential problem is minimized If high viscosity is observed it is important to reduce viscosity by either Diluting the sample or Including a DNA shear step in your protocol after the addition of Lysis Binding Buffer Use force to shear the DNA properly by passage through a syringe Repeated shearing causes foaming of the lysate due to a detergent in the buffer however this should not affect the mRNA yield If foam is observed centrifuge the sample for 30 seconds to reduce the foam No signal observed after RT PCR RNA is degraded by contaminating RNases Use RNase free pipette tips with aerosol barriers Change gloves frequently Clean pipettors with RNaseZap solution Inhibitors are present in the mRNA sample If performing direct isolation of mRNA from cells or tissues repeat washing steps from step 5 Remove the sample tube from the magnet and resuspend the Dynabeads mRNA complex in 100 uL Washing Buffer A by careful pipetting o
48. ubit RNA Assay Kit with the Qubit Fluorometer 2 Assess the quality of the mRNA a Run 1uL of the sample on an Agilent 2100 Bioanalyzer instrument with the Agilent RNA 6000 Nano Kit Follow the manufacturer s instructions for performing the assay b Using the 2100 expert software review the percent of 18s and 28s present in the sample Note For instructions on how to review the size distribution refer to the Agilent 2100 Bioanalyzer Expert User s Guide 1 8 ug Run 1 uL of the collected sample on an Agilent 2100 Bioanalyzer instrument using the Agilent RNA 6000 Pico Kit Follow the manufacturer s instructions to perform the assay Do not further quantitate your sample Note For instructions on how to review the size distribution refer to the Agilent 2100 Bioanalyzer Expert User s Guide Dynabeads mRNA DIRECT Micro Kit User Guide Chapter 3 mRNA Isolation from Purified Total RNA 3 mRNA isolation from 100 ng 1 ug total RNA samples mRNA isolation from 100 ng 1 pg total RNA samples Before you begin Read Guidelines for best results on page 26 If preparing RNA Seq whole transcriptome libraries from the mRNA prepare the Ion Total RNA Seq Kit v2 reagents as directed in the Ion Total RNA Seq Kit v2 User Guide Pub no 4476286 before beginning this mRNA isolation protocol Bring all the Dynabeads mRNA DIRECT Micro Kit reagents to room temperature except the 10 mM Tris HCl
49. ularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Handle chemical wastes in a fume hood Ensure use of primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage After emptying a waste container seal it with the cap provided Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Specific chemical p CAS Chemical Phrase handling 26628 22 8 Sodium Azide Sodium azide may react with lead and copper plumbing to form highly explosive metal azides 34 Dynabeads mRNA DIRECT Micro Kit User Guide Appendix D Safety Biological hazard safety Biological hazard safety A WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicab
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