SensationPlus FFPE Amplification and WT 1 Labeling Protocol

Transcription

1 SensationPlus FFPE Amplification and WT 1 Labeling Protocol Protocol performed using the SensationPlus FFPE Amplification and WT Labeling Kit ( rxn) Dilution of Poly-A Controls Requires the Affymetrix GeneChip Poly-A Control Kit (cat#900433) Note: Chill refrigerated plate centrifuge to 4 C Serial Dilution of Poly-A RNA control Stock Total RNA Input Serial Dilutions Volume of 4 th Dil. to Amount 1 st Dilution 2 nd Dilution 3 rd Dilution 4 th Dilution add to Total RNA 20 ng 1:20 1:50 1:50 1:50 2 ul 50 ng 1:20 1:50 1:50 1:20 2 ul 100 ng 1:20 1:50 1:50 1:10 2 ul Recommended 500 ng 1:20 1:50 1:50 1:5 2 ul Note: The first dilution of the Poly-A Controls can be stored for 6 weeks at -20 C and freeze-thawed 8x. 1. Add 2ul of Poly-A control Stock to 38ul Poly-a dilution buffer to make the First Dilution (1:20 dilution). Mix well and microcentrifuge. 2. Add 2ul of First Dilution to 98ul Poly-A dilution buffer to make Second Dilution (1:50 dilution). Mix well and microcentrifuge. 3. Add 2ul of Second Dilution to 98ul Poly-A dilution buffer to make Third Dilution (1:50 dilution). Mix well and microcentrifuge 4. Add 2ul of Third Dilution to 18ul Poly-A dilution buffer to make Fourth Dilution (1:10 dilution). Mix well and microcentrifuge. 5. Add 2ul of the Fourth Dilution to 100ng total RNA to make up the 5ul Total RNA/Poly-A RNA Control mix. First Strand cdna Synthesis 1. In an appropriately labeled 0.3 ml PCR plate, adjust the volume of 100ng of FFPE total RNA/Poly-A RNA Control mix to 7ul with nuclease free water. 2. On ice, add 4ul RT RNA-Primer Mix (red cap) to 7ul of the FFPE total RNA/Pol-A RNA Control to make an 11ul RNA-Primer mix. 3. Seal plate with Microseal B adhesive seal. 4. Incubate the RNA/Primer mix in a thermal cycler using the following program: SPRIME 80 C 10 min. Note: Use heated lid w/ 5 C tracking 5. Following the 2 min incubation at 4 C, transfer samples to ice.

2 6. Prepare Amplification RT Master Mix as follows for a single reaction on ice. Adjust as needed for multiple reactions. 2 RT Buffer Mix (red cap) 4ul DTT (red cap) 2ul dntp mix (red cap) 1ul RT Enzyme Mix (red cap) RNase Inhibitor (red cap) 1ul 1ul Total 9ul Gently mix Do Not Vortex 7. Centrifuge plate briefly at 4 C. Remove seal and place on ice. 8. Transfer 9ul of Amplification RT Master Mix to each reaction for 20ul total. 9. Mix well by pipetting. Seal plate with Microseal B adhesive seal. 10. Incubate the RT reaction in a thermal cycler using the following program: S*RT 42 C 1 hr 25 C 2 min. Note: Use heated lid w/ 5 C tracking NOTE: Shake the RNA Purification Beads to fully resuspend. Aliquot the appropriate amount of beads and keep at RT (36ul beads/reaction is required). 11. Immediately proceed to cdna Purification when the 25 C incubation is complete. Purification of cdna with RNA Purification Beads Note: Use RNA Purification Beads Amplification. Mix well and remove needed amount to RT. Note: Prepare fresh dilutions of ethanol each time RNA Purification Beads are used. Note: Preheat nuclease-free water at 65 C for elution. 1. Mix the aliquot of RNA Purification Beads- Amplification and ensure complete resuspension. 2. Remove adhesive seal. 3. Add 36ul of RNA Purification Beads to each reaction and mix well by pipetting 10-20x. 4. Add 30ul of Absolute ETOH and mix well by pipetting 10-20x. Note: Do not add ETOH to non-ffpe samples. 5. Incubate for 10 min. at RT (20-25 C). 6. Place the plate on the magnetic stand 10 min. to separate the beads from the solution. 7. Slowly aspirate and discard the cleared solution DO NOT DISTURB BEADS 8. Keeping plate on the magnetic stand, add 180ul of 70% ETOH to each well and incubate for 30 sec. at RT. 9. With a pipette set to 200ul slowly remove the ETOH and discard DO NOT DISTURB BEADS 10. Repeat the ETOH wash 2x for a total of three washes. Completely remove the final ETOH wash. 11. Air dry the bead pellets on the magnetic stand for 5 min. Pellet will appear shiny with no liquid left. Note: Under-drying or over-drying the bead pellet may result in reduced recoveries.

3 12. Remove the plate from the magnetic stand. 13. Add 14ul of the 65 C Nuclease Free H 2 O to each well. Once H 2 O is added to all wells incubate at RT (20-25 C) for 3 min. While incubating at RT resuspend the beads by pipetting up and down. 14. Place the plate on the magnetic stand for 3 min. to separate the beads from solution. 15. Carefully transfer 12ul clarified solution to a new labeled 0.3ml PCR plate. DO NOT DISTURB BEADS. 16. Measure volume of transferred samples and adjust to 12ul with nuclease free H 2 O, if necessary. 17. Seal plate with Microseal B adhesive seal. Note: A minimal amount of bead carryover will NOT inhibit subsequent enzymatic reactions. 18. Immediately proceed to Promoter Synthesis. 3 Promoter Synthesis 1. Heat 12ul purified cdna plate using the following cycling profile: P1 80 C 10 min. Note: Use heated lid w/ 5 C tracking 2. Transfer plate to ice after 4 C incubation is complete. 3. Prepare Tailing Master Mix as follows for a single reaction on ice. Adjust as needed for multiple reactions. Tailing Buffer Mix (blue cap) 6ul Tailing Enzyme Mix (blue cap) 2ul Total 8ul 4. Gently tap to mix Tailing Master Mix and centrifuge briefly. 5. Centrifuge plate at 4 C briefly and remove adhesive seal. Place on ice. 6. Add 8ul of Tailing Master Mix to 12ul of purified cdna for a final volume of 20ul. Pipette to mix. 7. Seal plate with Microseal B adhesive seal. 8. Incubate the 20ul Tailing Reaction using the following profile: TAIL 37 C 2 min. Note: Use heated lid w/ 5 C tracking 80 C 10 min. 9. Transfer plate to ice. 10. Prepare the Promoter Synthesis Master Mix as follows on ice. Adjust as needed for multiple reactions. Promoter Synthesis Buffer Mix (green cap) 4ul Promoter Synthesis Enzyme Mix (green cap) 1ul Total 5ul 11. Gently tap to mix Promoter Synthesis Master Mix and centrifuge briefly. 12. Centrifuge plate briefly at 4 C and remove the adhesive seal. Place on ice.

4 4 13. Add 5ul of Promoter Synthesis Master Mix to each sample for a 25ul final volume. Pipette to mix. 14. Seal plate with a Microseal B adhesive seal. 15. Incubate the 25ul Promoter Synthesis Reaction using the following profile: PSR 25 C 30 min. Note: Use heated lid w/ 5 C tracking 16. Transfer plate to RT. 17. Immediately proceed to In Vitro Transcription. In Vitro Transcription Note: Thaw the T7 Nucleotide Mix (yellow cap) and T7 Buffer Mix (yellow cap) at RT and keep at RT until use. Thoroughly vortex the T7 Buffer mix to fully resuspend. Microfuge briefly. 1. Prepare IVT Master Mix as follows at RT. Adjust as needed for multiple reactions. T7 Nucleotide Mix (yellow cap) 16ul T7 Buffer Mix (yellow cap) 5ul T7 Enzyme mix (yellow cap) 9ul Total 30ul 2. Gently mix the IVT Master Mix by tapping and centrifuge briefly. 3. Remove plate seal and add 30ul of IVT Master Mix to the 25ul Promoter modified cdna for a final volume of 55ul. Pipette to mix. 4. Seal plate with Microseal B adhesive seal. 5. Incubate the 55ul IVT reaction using the following profile: IVT 37 C 18 hours Note: Use heated lid w/ 5 C tracking 4 C hold 6. Proceed to Purification of SenseRNA with RNA Purification Beads. Safe Stopping Point: If purification is not initiated, sealed plate can be store at -20 C. Purification of senserna with RNA Purification Beads Note: Use RNA Purification Beads Amplification. Mix well and remove needed amount to RT. Note: Prepare fresh dilutions of ETOH each time RNA Purification Beads are used. Note: Preheat the nuclease-free H 2 O at 65 C for elution. 1. Mix the aliquot of RNA Purification Beads-Amplification and ensure complete resuspension. 2. If plate was stored at -20 C, warm to RT before proceeding with the bead purification. 3. Remove adhesive seal. 4. Add 99ul of RNA Purification beads to each well. Mix by pipetting 10-20x. 5. Incubate for 10 min. at RT (20-25 C).

5 6. Place the plate on the magnetic stand 10 min. to separate the beads from the solution Slowly aspirate and discard the cleared solution DO NOT DISTURB BEADS 8. Keeping plate on the magnetic stand, add 180ul of 70% ETOH to each well and incubate for 30 sec. at RT. 9. With a pipette set to 200ul slowly remove the ETOH and discard DO NOT DISTURB BEADS 10. Repeat the ETOH wash 2x s for a total of three washes. Completely remove the final ETOH wash. 11. Air dry the bead pellets on the magnetic stand for 5 min. Pellet will appear shiny with no liquid left. Note: Under-drying or over-drying the bead pellet may result in reduced recoveries. 12. Remove the plate from the magnetic stand. 13. Add 23ul of the 65 C Nuclease Free H 2 O to each well. Once H 2 O is added to all wells incubate at RT (20-25 C) for 3 min. While incubating at RT resuspend the beads by pipetting up and down. 14. Place the plate on the magnetic stand for 3 min. to separate the beads from solution. 15. Carefully transfer clarified solution to a new labeled 0.3ml PCR plate. DO NOT DISTURB BEADS. Note: When SenseRNA is very concentrated it may be difficult to aspirate because the beads quickly aspirate as well. When it is difficult to aspirate the purified senserna, remove the plate from the magnetic stand and add an additional 10-20ul of 65 C nuclease-free H 2 O to each well that is difficult to aspirate. Mix ell by pipetting until fully resuspended and incubate at RT (20-25 C) for 1 min. to elute the sample. Proceed to step 14 above. Minimal Bead carryover will not inhibit subsequent enzymatic reactions. 16. Measure volume of purified senserna and adjust to 21ul 17. Perform NanoDrop assay on 1ul of the sense RNA. Record concentration and 260/280 ratio for each sample. Note: In general, the yield of senserna from FFPE samples does not require dilution. Safe Stopping Point: Sealed plate can be store at -20 C. Labeling Procedure Double - Stranded cdna Synthesis Note: 25ug senserna (in a volume of 20ul) is recommended for the labeling procedure. If 25ug senserna is unavailable, proceed with 10-25ng of senserna. For each sample use as much senserna as possible, up to 25ug but not less than 10ug. 1. Using a new labeled 0.3ml PCR plate on ice, transfer 25ug of each senserna and adjust the volume to 20ul with nuclease-free H 2 O. 2. On ice, add 3ul WT Labeling Primer Mix (red cap) to the 20ul senserna for a total of 23ul. 3. Seal plate with Microseal B adhesive seal. 4. Incubate the 23ul senserna-primer Mix using the following profile: P2 80 C 5 min. Note: Use heated lid w/ 5 C tracking 5. After 2 min. incubation at 4 C transfer the plate to ice.

6 6. Prepare the Labeling RT Master Mix as follows on ice. Adjust as need for multiple reactions. 6 RT Buffer Mix (red cap) 8ul DTT (red cap) 4ul Labeling dntp Mix (red cap) 2ul RT Enzyme Mix (red cap) 2ul RNase Inhibitor (red cap) 1ul Total 17ul 7. Gently mix the Labeling RT Master Mix by tapping and centrifuge briefly. 8. Remove plate seal and add 17ul of Labeling RT Master Mix to the 23ul senserna-primer mix for a final volume of 40ul. Pipette to mix. 9. Seal plate with a Microseal B adhesive seal. 10. Incubate the 40ul reaction for First Strand Synthesis using the following profile: RT1 42 C 120 min Note: Use heated lid w/ 5 C tracking 25 C 2 min 11. After the 2 min 25 C incubation, transfer plate to ice. 12. Prepare the Second Strand Master Mix as follows on ice. Adjust as needed for multiple reactions. MgCl 2 (purple cap) 13ul Labeling dntp Mix (red cap) 2ul Klenow (purple cap) 3ul RNase H (purple cap) 2ul Total 20ul 13. Gently mix the Second Strand Master Mix by tapping and centrifuge briefly. 14. Remove plate seal and add 20ul of Second Strand Master Mix to the 40ul First Strand Reaction for a final volume of 60ul. Pipette to mix. 15. Incubate the 60ul Second Strand Reaction using the following profile: 2ND 37 C 40 min. Note: Use heated lid w/ 5 C tracking 75 C 10 min. 16. After 2 min. incubation at 4 C, transfer plate to RT, remove seal and immediately add 2ul of WT Stop Solution (brown cap) to each sample for a total volume of 62ul. After adding WT Stop Solution to each sample mix by pipetting at a 45ul volume setting. 17. Incubate the 62ul Stop Reaction using the following profile: RT2 65 C 30 min. Note: Use heated lid w/ 5 C tracking 25 C 2 min. 18. After 2 min. incubation at 25 C, transfer plate to RT, remove plate seal and immediately add 8ul of WT Neutralization Mix (brown cap) to each sample for a total volume of 70ul. After adding WT Neutralization Mix to each sample mix by pipetting at a 55ul volume setting. Safe Stopping Point: Sealed plate can be store at -20 C.

7 7 Purification of Double-Stranded cdna for Labeling with RNA Purification Beads Note: Use RNA Purification Beads Labeling. Note: Prepare fresh dilution of 70% ETOH. Note: Preheat the nuclease-free H 2 O at 65 C for elution. 1. Mix the RNA Purification Beads Labeling until fully resuspended. Remove aliquot of the appropriate volume and return main stock of beads to 4 C. 2. Make sure samples are at RT before initiating bead purification. 3. For each reaction add 126ul of RNA Purification Beads Labeling to each sample and mix well by pipetting 10-20x. 4. Add 65ul of RT 100% ETOH to each sample and mix well by pipetting 10-20x. 5. Incubate at RT 10 min. 6. Place the plate on the magnetic stand for 10 min. to fully separate beads from the solution. 7. Carefully aspirate and discard the cleared solution. DO NOT DISTURB BEADS. 8. While on the magnetic stand add 170ul of 70% ETOH to each well and incubate for 30 sec. at RT 9. Using a pipette set to 200ul, slowly aspirate the ETOH wash solution and discard. DO NOT DISTURB BEADS. 10. Repeat ETOH wash 2x s for a total of three washes. Completely remove the final wash solution. 11. Air dry bead pellets on the magnetic stand for 5 min. Pellet will appear shiny with no liquid left. Note: Under-drying or over-drying the bead pellet may result in reduced recoveries. 12. Remove plate from magnetic stand. 13 Add 25ul of the 65 C preheated nuclease free H 2 O to each well. Once H 2 O is added to all wells incubate at RT (20-25 C) for 3 min. While incubating at RT gently resuspend the beads by pipetting up and down. 14. Place the plate on the magnetic stand for 3 min. to separate the beads from the solution. 15. Slowly aspirate the purified double-stranded cdna and transfer to a new appropriately labeled 0.3ml PCR plate. Be careful not to disturb beads. Note: Minimal Bead carryover will not inhibit subsequent enzymatic reaction. 16. Perform a Nanodrop assay of the purified cdna. Record yields. Double-Stranded cdna Amounts Required for Terminal Labeling 49-Format Array 100-Format Array 169-Format Array ds-cdna per Array 6 ug 5 ug 3.5 ug Minimum Conc. of Elute 270 ng/ul 225 ng/ul 156 ng/ul Safe Stopping Point: Seal plate with Microseal B and store at -20 C.

8 Terminal Labeling of cdna 8 Note: The minimum amount of purified double-stranded cdna that is required for the terminal labeling reaction by array format is provided in the proceeding table. Seal and retain unused doudlestranded cdna at -20 C. 1. Transfer an adequate volume of each sample to obtain the necessary quantity of double-stranded cdna into a new 0.3ml PCR plate. Adjust the ds cdna volume to 22.5ul with nuclease free H 2 O. 2. Prepare the Terminal Labeling Reaction Master Mix as follows on ice. Adjust as need for multiple reactions. Fragment and Label Buffer Mix (purple cap) Fragment and Label Enzyme Mix (purple cap) 6ul 1.5ul Total 7.5ul 3. Gently tap to mix and briefly centrifuge Terminal Labeling Reaction Master Mix. 4. Add 7.5ul of Terminal Labeling Reaction Master Mix to each 22.5ul ds cdna for a final volume of 30ul. Mix by pipetting. 5. Seal plate with Microseal B adhesive seal. 6. Incubate the 30ul Terminal Labeling Reaction using the following profile: LABEL 37 C 60 min. Note: Use heated lid w/ 5 C tracking 93 C 2 min. 7. Proceed to WT expression array hybridization.

9 Whole-Transcript Expression Array Hybridization 9 This procedure requires the use of the GeneChip Hybridization, Wash and Stain kit (Affymetrix ). Note: Prewarm the hybridization oven to 47 C. Note: Prewarm arrays to RTº ~1 hour before use and label appropriately. Enter experiment info and bar codes into Command Console. Note: Denature the 20x Eukaryotic Hybridization Controls at 65 C for 5 min before Hybridization Cocktail set up. 1. Prepare the hybridization cocktail in appropriately 1.5ml tubes as follows (adjusted for sample number): Note: Verify the array format being used. Vol. for 1 Vol. for 1 Vol. for 1 Component 49 format 100 format 169 format Fragmented and Labeled ds cdna Target 30ul 30ul 30ul Control Oligo B2 (3nM) 3.3ul 2.2ul 1.5ul 20x Eukaryotic Hyb Controls 10ul 6.5ul 4.5ul 2x Hybridization Mix 100ul 65ul 45ul DMSO 20ul 13ul 9ul Nuclease Free Water 36.7ul 13.3ul 0ul Total Volume 200ul 130.0ul 90ul 2. Low speed vortex briefly and spin down. 3. Denature cocktail for 5 min. at 99ºC in heat block. 4. Cool for 5 min. at 47ºC in the Hybridization Oven 640. Microcentrifuge at full speed 1 min. 5. Inject the appropriate amount of denatured cocktail into the array. Seal septa with Tough Spots. Array Hyb. Format Volume ul ul ul 6. Place arrays in the Hybridization oven 640 and hybridize at 47ºC, 60rpm, for 17 hours ± 1 hour. 7. Prepared staining reagents as indicated below. 8. Prewarm the necessary amount of Wash A and Wash B overnight at RT protected from light.

10 Array Washing and Staining 10 This procedure requires the use of the GeneChip Hybridization, Wash and Stain kit (Affymetrix ). Note: Staining reagents can be aliquoted and stored at 4 C overnight prior for the Wash and Stain procedure (volumes are sufficient for one array): a. remove Stain cocktail 1, Stain cocktail 2, and Array Holding buffer from Stain Module box 1. b. Gently tap bottles to mix well. c. Aliquot the following reagents: i. 600ul Stain Cocktail 1 into a labeled 1.5ml amber tube Fluidics Position 1 ii. 600ul Stain Cocktail 2 into a labeled 1.5ml clear tube Fluidics Position 2 iii. 800ul Array Holding Buffer into a labeled 1.5ml clear tube Fluidics Position 3 iv. Centrifuge tubes if necessary. 1. Verify that experiment files and GeneChip barcodes have been entered into Command Console. 2. Prime the necessary Fluidics station 450 s. Ensure that the appropriate Wash A and Wash B buffers have been installed in there appropriate location. 3. After 17 hours ± 1 hour of hybridization remove arrays and remove hyb. cocktail to the original tube and store at -80ºC 4. Refill probe array with RT Wash A buffer completely. Note: If necessary the probe arrays can be stored at 4ºC for up to 3 hours proceeding the washing and staining fluidics procedure. 5. Initiate fluidics profile. Follow fluidics station prompts to install array, the Stain Cocktails and Array Holding Buffer on the fluidics Station. 49 Format 100 Format 169 Format Fluidics Profile Used FS450_0001 FS450_0002 FS When fluidics is complete, remove arrays and check for bubbles. If bubbles are present manually fill array with RT Array Holding Buffer. Place arrays at RT protected from light until ready to scan. 7. Engage wash blocks so that modules can prime. Follow prompts on fluidics monitors. 8. If all arrays are complete with fluidics, perform the fluidics shutdown protocol using Shutdown_ Power down units when shutdown is complete. Scanning NOTE: Initiate scanner when the fluidics stations are initiated. If forgotten Allow the scanner to warm a minimum of 15 min prior to initiating a scan. 1. Clean array glass with water and a kim wipe. DO NOT use alcohol. 2. Install arrays into carousel of scanner starting with position 1 marked in red. Close scanner. 3. Select the start scan icon from AGCC. Select the appropriate option concerning array temperature. 4. Initiate run using a single scan. Do not allow rescans. 5. When scans are complete review for grid alignment and artifacts.