Protocol




Overview

Protocol: Transcript Capture and Ligation Mediated Amplification

In reverse transcription, mRNA transcripts from whole cell lysate or purified RNA are isolated by capturing the polyA tail of the transcript on the surface of a polyT coated turbocapture plate. Then the reverse transcriptase enzyme is used to build a cDNA complement to this polyT tail. Upstream and downstream probes containing gene specific, LUA “barcode” tags, and universal primer sequences are then added to the mix. The probes anneal and ligate to the gene specific region of the transcript. Finally, universal biotinylated primers are added to the mix and PCR amplified. The final product is biotinylated, barcoded PCR amplicon of 104-nt length.

Transcript Capture (CAP)

Purpose

Immobilize mRNA from lysed cells onto the walls of the Turbocapture plate, by providing a dT coated surface which binds the poly-A tail of each mRNA transcript.

RELATED PROTOCOL 
This step requires materials produced via the related protocol Cell Based Assay. Alternately, mRNA acquired by some other method can be used.
IMPORTATNT
Prepare materials and assets in pre-PCR and RNAse-free environment.

Preparation

  1. Thaw LYSIS plates at 4°C
  2. Thaw Reference Total RNA M07 on ice
  3. Prepare CyBio tips according to plate layout for lysis transfer
  4. Label Turbocapture plates

Materials

  • TCL buffer M01
  • MicroAmp seals M02
  • CyBio 384-well tips M03
  • Turbocapture plates M04
  • 1.5ml eppendorf tubes M05

Assets

  • Vortex A12
  • Benchtop centrifuge A09
  • CyBio Vario 384 A01
  • Matrix electronic pipette A13

Reagent Mix

ID Name Step Composition Volume/Well Use
MIX01 MCF7 Total RNA master mix CAP 19.7μl TCL Lysis Buffer M01
0.3μl MCF7 Total RNA M07
20μl Keep on ice, incubate at room temp for 5m before using

Procedure

  1. Label each new Turbocapture plate with information listed on the corresponding cell lysate
    source plate (compound set, cell line, date, plate #). Include also current date and initials.

  2. Document names of plates being taken through RT on tracking sheet.

  3. Centrifuge thawed cell lysate (LYSIS) plates for 1min @1000RPM. Keep plates on ice until ready to transfer.

  4. Place LYSIS plate at Position 1 of CyBio and its corresponding labeled Turbocapture plate (CAP) at Position 2.

    TIP: Check orientation of CAP and LYSIS plates.

  5. Load CyBio 384 tip cartridge.

    TIP: Ensure CyBio tips have been arranged in rack to transfer only relevant lysate from LYSIS plate as specified by the ‘Plate Format’. Take care not to let any lysate get into control wells.

  6. Transfer 20 μl lysate from LYSIS to CAP plate using CyBio program: 384 Lysate Capture .

  7. Repeat steps 1-5 until all LYSIS plates have been transferred.

  8. Prepare Reference Total RNA controls.

    1. Mix Total RNA master mix MIX01 for each cell line included in ‘Plate Format’, using 1.5 ml Eppendorf tubes. Flick Eppendorf tubes to mix and incubate at room temperature for 5 min.
      IMPORTANT: Keep total RNA on ice, limit freeze/thaw cycles to 4, and return to -80C storage as soon as possible. Do not try to mix directly in wells.
    2. Document REF RNAs prepared and their F/T# on tracking sheet.
    3. Pipette 20μl of Total RNA master mix to corresponding control wells of all CAP plates
      according to ‘Plate Format’.
  9. Seal plate with MicroAmp seal.

  10. Centrifuge plates for 15 seconds at 1000 RPM to settle liquid in wells.

    TIP: Check for empty or low volume wells and mark on tracking sheet.

  11. Incubate at Room Temperature for 1 hour.

  12. Proceed directly to preparation for Reverse Transcription (FIRST).

Reverse Transcription (FIRST)

Purpose

Synthesize an immobolized cDNA that is complementary to the mRNA by using the immobilized dT as the primer and the captured mRNA as the template.

IMPORTANT: Prepare materials and assets in Pre-PCR and RNAse-Free environment.

Preparation

  1. Thaw 5x M-MLV RT buffer M18
  2. Thaw 25mM dNTPs M19
  3. Leave M-MLV RT Enzyme M20 at -20°C

Materials

  • Plates from CAP step
  • DEPC treated water M17 kept at -20°
  • CyBio 384-well tips M03
  • Reagent reservoir M16
  • Super rags M12
  • Aluminum foil M13
  • MicroAmp seal M02

Assets

  • Benchtop centrifuge A09
  • CyBio Vario 384 A01
  • Matrix electronic pipette A13
  • PrePCR thermocyclers A02
  • 20° freezer, 4th floor A04

Reagent Mix

ID Name Step Composition Volume/Well Use
MIX02 RT master mix RT 3.7μl DEPC treated water M17
1μl 5X M-MLV RT Buffer M18
0.1μl 25mM dNTPs M19
0.2μl M-MLV RT enzyme M20
5μl Keep on ice, add enzyme when ready to begin master mix addition

Procedure

  1. Prepare FIRST master mix without enzyme MIX02 on ice
    IMPORTANT: Keep enzyme at -20°C and add just before beginning procedure. Complete as much of procedure as possible on ice.

    1. Invert several times to mix and pour master mix into reagent reservoir on ice.

  2. Spin out lysate from CAP plates
    1. Remove centrifuge plate holders from rotor.
    2. Remove seal of CAP plate.
    3. Fold Super Rag in half and place on top of plate.
    4. Wrap plate in aluminum foil to reduce liquid spillage into centrifuge.
    5. Invert plate.
    6. Place wrapped plate inverted into plate holder.
    7. Spin for 1 min at 1000RPM to remove liquid from wells.
  3. Transfer 5μl of master mix from the reservoir to destination CAP plate using CyBio program: 5ul Reagent Addition.
    1. Load new CyBio 384 tip cartridge.
      TIP: Use the same tips for all transfers in this step.
    2. Load CAP plate in Position 1 and place RT master mix source plate in Position 2.
    3. Run CyBio program.
    4. Repeat until all CAP plates have been processed.
      TIP: Do not let the CAP plates sit dry for too long. Do not use plate stacker. Additional plates can be spun out while running the CyBio.
  4. Seal plates well with MicroAmp seal.
  5. Centrifuge plates for 15 seconds at 1000 RPM to settle liquid in wells.
    TIP: Check for debris in wells and mark on tracking sheet.<.pre>
  6. Incubate in pre-PCR thermocycler using program: 37for90.
    STOP POINT: If procedure will be continued at a later time, store CAP plates at -20°C, sealed tightly with aluminum foil or MicroAmp seals. Be sure to note the date Reverse Transcription was completed.
  7. Proceed directly to preparation for Probe Anneal (ANNL).

Probe Anneal (ANNL)

Purpose

Bind probe pairs targeting specific sequences for genes of interest to target cDNA which corresponds to them. These sequences are located adjacent to each other on the cDNA template and contain LUA tags and universal primer sites to be used in later steps.

IMPORTANT: Prepare materials and assets in Pre-PCR and RNAse-Free environment.

Preparation

  1. Thaw 10X DNA ligase buffer M23
  2. Thaw probe pool M22 at room temperature
  3. Check FIRST plates for evaporated wells

Materials

  • Plates from FIRST step
  • CyBio 384-well tips M03
  • Nuclease free water M21
  • Reagent reservoir M16
  • Super rags M12
  • Aluminum foil M13
  • MicroAmp seal M02

Assets

  • Vortex A12
  • Plate heater A30
  • Benchtop centrifuge A09
  • CyBio Variable 384 A01
  • Matrix electronic pipette A13
  • Pre PCR thermocycler A02
  • 20°C freezer, 4th floor A04

Reagent Mix

ID Name Step Composition Volume/Well Use
MIX03 Probe anneal master mix ANNL 4.22μl Nuclease free water M21
0.28μl "1000" probe pair mix M22
0.5μl 10X Taq ligase buffer M23
5μl Keep at RT, warm probes before adding

Procedure

  1. Prepare ANNL master mix without probes MIX03 at room temperature.
    IMPORTANT: Add probes just before beginning procedure.
    TIP:Warm probes to 50 C for 10 to 20 seconds before adding.
    1. Vortex to mix and pour into reagent reservoir at room temperature.
  2. Spin out master mix from FIRST plates
    1. Remove centrifuge plate holders from rotor.
    2. Remove seal of FIRST plate.
    3. Fold Super Rag in half and place on top of plate.
    4. Wrap plate in aluminum foil to reduce liquid spillage into centrifuge.
    5. Invert plate.
    6. Place wrapped plate inverted into plate holder.
    7. Spin for 1 min at 1000RPM to remove liquid from wells.
      TIP: Inspect plates for debris, liquid, etc. in centrifuge.
  3. Transfer 5μl of master mix from reservoir to destination FIRST plate using CyBio program 5ul Reagent Addition.
    1. Load new CyBio 384 tip cartridge.
      TIP: Use the same tips for all transfers in this step.
    2. Load FIRST plate in Position 1 and load Probe Anneal master mix in Position 2.
    3. Run CyBio program
    4. Repeat until all FIRST plates have been processed.
      TIP:Do not let the FIRST plates sit dry for too long. Additional plates can be spun out while running the CyBio.
  4. Seal plates well with MicroAmp seal.
    IMPORTANT: Make a tight seal by pressing firmly and edging the seal with a pen.
  5. Centrifuge plates for 15 seconds at 1000 RPM to settle liquid in wells.
    TIP: Check for debris in wells and mark on tracking sheet.
  6. Incubate overnight in Pre PCR thermocycler using program probebind6hramp.
    STOP POINT: If procedure will be continued at a later time, store ANNL plates at -20°C in the 4th floor freezer A04, sealed tightly with aluminum foil or MicroAmp seals. Be sure to note the date Probe Anneal was completed.
  7. Proceed directly to preparation for Probe Ligation (LIG).

Probe Ligation (LIG)

Purpose

Ligate together the two probes of each probe pair to form a single strand comprised of the entire 40 nucleotide gene specific sequence flanked by a LUA tag and T3 & T7 primer site.

IMPORTANT:Prepare materials and assets in Pre-PCR and RNAse-Free environment.

Preparation

  1. Thaw 10X Taq DNA ligase buffer M23
  2. Check ANNL plates for evaporated wells
  3. Note hold duration of ANNL plates on tracking sheet

Materials

  • Plates from ANNL step
  • CyBio 384-well tips M03
  • Nuclease free water M21
  • Taq DNA ligase enzyme M20 kept at -20°C
  • Reagent reservoir M16
  • Super rags M12
  • Aluminum foil M13
  • MicroAmp seal M02

Assets

  • Vortex A12
  • Plate heater A30
  • Benchtop centrifuge A09
  • CyBio Variable 384 A01
  • Matrix electronic pipette A13
  • Pre PCR thermocycler A02
  • 20°C freezer, 4th floor A04

Reagent Mix

ID Name Step Composition Volume/Well Use
MIX04 Ligation master mix LIG 4.4375μl Nuclease free water M21
0.5μl 10X Taq ligase buffer M23
0.0625μl Taq DNA ligase M24
5μl Keep on ice, add enzyme when ready to begin master mix addition

Procedure

  1. Prepare LIG master mix without enzyme MIX04 on ice.
    IMPORTANT:Keep enzyme at -20 C and add just before beginning procedure. Complete as much of procedure as possible on ice.
    1. Invert several times to mix and pour master mix into reagent reservoir on ice.
  2. Spin out master mix from ANNL plates
    1. Remove centrifuge plate holders from rotor.
    2. Remove seal of ANNL plate.
    3. Fold Super Rag in half and place on top of plate.
    4. Wrap plate in aluminum foil to reduce liquid spillage into centrifuge.
    5. Invert plate.
    6. Place wrapped plate inverted into plate holder.
    7. Spin for 1 min at 1000RPM to remove liquid from wells.
      TIP: Inspect plates for debris, liquid, etc. in centrifuge.
  3. Transfer 5μl of master mix from reservoir to destination ANNL plate using CyBio program 5ul Reagent Addition.
    1. Load new CyBio 384 tip cartridge.
      TIP: Use the same tips for all transfers in this step.
    2. Load ANNL plate in Position 1 and load Probe Anneal master mix in Position 2.
    3. Run CyBio program
    4. Repeat until all ANNL plates have been processed.
      TIP:Do not let the ANNL plates sit dry for too long. Additional plates can be spun out while running the CyBio.
  4. Seal plates well with MicroAmp seal.
    IMPORTANT: Make a tight seal by pressing firmly and edging the seal with a pen.
  5. Centrifuge plates for 15 seconds at 1000 RPM to settle liquid in wells.
    TIP: Check for debris in wells and mark on tracking sheet.
  6. Incubate overnight in Pre PCR thermocycler using program: probelig384.
    TIP:The program holds at 45°C for one hour, 65°C for 10 minutes, then holds overnight at 4°C.
    STOP POINT: If procedure will be continued at a later time, store LIG plates at -20°C in the 4th floor freezer A04 , sealed tightly with aluminum foil or MicroAmp seals. Be sure to note the date Probe Ligation was completed.
  7. Proceed directly to preparation for PCR Amplification step (PCR).

PCR Amplification (PCR)

Purpose

Denature ligated probe pairs from their cDNA templates, and amplify gene transcripts of interest exponentially from their cT3 and cT7 primer sites. The final products are same length, biotinylated, barcoded PCR amplicons.

IMPORTANT:Prepare materials and assets in Pre-PCR and RNAse-Free environment.

Preparation

  1. Thaw 25 mM dNTPs M19 with T3 M27 & T7 M38 primers
  2. Thaw 25mM MgCl2 M26
  3. Thaw 10X PCR buffer M25
  4. Check LIG plates for evaporated wells

Materials

  • Plates from LIG step
  • Nuclease free water M21
  • HotStarTaq M29 kept at -20°C
  • CyBio 384 well tips M03
  • Reagent reservoir M16
  • Super rag M12
  • Aluminum foil M13
  • MicroAmp seal M02

Assets

  • Benchtop centrifuge A09
  • CyBio Vario 384 A01
  • Matrix electronic pipette A13
  • Pre PCR thermocycler A02
  • 20°C freezer, 4th floor A04

Reagent Mix

ID Name Step Composition Volume/Well Use
MIX05 PCR master mix PCR 12.768μl Nuclease free water M21
1.5μl 10X PCR buffer M25
0.51μl MgCl2 M24
0.096μl 25mM dNTPs M19
0.015μl T3 primer 100μM M27
0.015μl T7 primer 100μM M28
0.096μl HotStarTaq M29
15μl Keep on ice, add enzyme when ready to begin master mix addition

Procedure

1.Prepare PCR master mix without enzyme MIX05 on ice.

IMPORTANT:Keep enzyme at -20°C and add just before beginning procedure. Complete as much of procedure as possible on ice.

1. Invert several times to mix and pour master mix into reagent reservoir on ice.
2. Spin out LIG plates.
1. Remove centrifuge plate holders from rotor.
2. Remove seal of LIG plate.
3. Fold Super Rag in half and place on top of plate.
4. Wrap plate in aluminum foil to reduce liquid spillage into centrifuge.
5. Place wrapped plate inverted into plate holder.

TIP:See Figure 5 for configuration of plate ready to spin out.

6. Spin for 1min at 1000RPM to remove liquid from wells.

TIP: Inspect plates for debris, liquid, etc. in centrifuge.

3. Transfer 15 μl of master mix from source plate to destination PCR plate using CyBio program 15 μl PCR addition.
1. Load new CyBio 384 tip cartridge

TIP: Use the same tips for all transfers in this step.

2. Load LIG plate in position 1 and place PCR master mix reagent reservoir in position 2.
3. Run CyBio program
4. Repeat until all LIG plates have been processed
4. Seal the plates with MicroAmp seal

IMPORTANT: Must seal plates very tightly before cycling. Press down firmly and edge wells tightly with a pen.

5. Centrifuge plates for 15 seconds at 1000RPM to settle liquid in wells

TIP:Check for debris in wells and mark on tracking sheet.

6. Incubate in 7th floor Post PCR Thermocycler using program PCR29 ALT 1M CYC.

TIP: The program brings the material to 92°C, then does 29 cycles as follows: 92°C for 1 minute, 60°C for one minute, 72°C for one minute. After cycling, it holds for 5 minutes at 72°C, ad then holds at 10°C.
STOP POINT: If procedure will be continued at a later time, store PCR plates at -20°C in the 7th floor freezer A05, sealed tightly with aluminum foil or MicroAmp seals. Be sure to
note the date PCR Amplification was completed.

7. Proceed directly to preparation for Hybridization (HYB)

Hybridization (HYB)

Purpose

Bind each amplified gene transcript to its corresponding Flexmap tag encoded bead in a detection plate. The tag-duo version of this protocol combines the 2 sets of 500 fluorescent beads at different proportions to create a single 1000-plex detection set instead of maintaining two separate subsets.

RELATED PROTOCOL: This step requires materials produced via the related protocol Bead Preparation.
IMPORTANT: Prepare materials and assets in a Post-PCR environment.

Preparation

  1. Thaw positive amplicon M74
  2. Check PCR plates for evaporated wells

Materials

  • Plates from PCR step
  • Coupled magnetic beads set A M32A
  • Coupled magnetic beads set B M32B
  • 1.5X TMAC MIX09
  • Reagent reservoir M16
  • Nanoscreen tips M30
  • Pink microseal M34
  • TE buffer M63

Assets

  • 7th floor benchtop centrifuge A10
  • 7th floor sonicator A17
  • 7th floor vortex A16
  • Biomek A27
  • Matrix electronic pipette A14
  • Post PCR thermocycler A03

Reagent Mixes

ID Name Step Composition Volume/Well Use
MIX07 Coupled bead mix HYB M32A Bead set A (includes controls at 0.5X)
M32B Bead set B (includes controls at 0.5X)
MIX09 1.5X TMAC
21μl Store at 4°C
MIX08 1X TMAC HYB 150ml TMAC M31
1.25ml 20% Sarkosyl M33
12.5ml Tris-HCl M35
2.0ml 0.5M EDTA M36
84.25ml DDW M40
250ml Store at room temperature
MIX09 1.5X TMAC HYB 225ml TMAC M31
1.88ml 20% Sarkosyl M33
18.75ml Tris-HCl M35
3ml 0.5M EDTA M36
1.37ml DDW M40
250ml Store at room temperature

Procedure

  1. Label each new HYB plate with information listed on the corresponding PCR source plate. Include also current date and initials.
  2. Document names of plates being taken through hybridization on tracking sheet.
  3. Prepare Coupled Bead Mix MIX07.
    TIP:Sonicate and vortex beads to ensure uniform dispersal.
  4. Add 21μl bead mix into plates using Biomek program cmap prep/ bead mix addition.
  5. Reconfigure Biomek deck for PCR amplicon transfer.
  6. Run program.
  7. Add positive amplicon control
    1. Dispense 5μl of positive amplicon into well B01
  8. Seal plates tightly with Microseal A.
  9. Incubate overnight at 45°C in 7th floor Post PCR Thermocycler using program hybridize.
    TIP: This program includes a 95C denaturing step before the 45 C hold.
  10. Proceed directly to preparation for SAPE Stain (SAPE).

Sape Stain (SAPE)

Purpose

Add a fluorescent label to each amplified gene bound to a given bead. This will allow the Luminex Flexmap3D system to quantitatively measure the amount of amplicon for each gene.

IMPORTANT:Prepare materials and assets in a Post-PCR environment.

Preparation

  1. Check HYB plates for evaporated wells
  2. Ensure Biomek waste is empty and DDW source is full

Materials

  • Plates from HYB step
  • DDW M40
  • 70% EtOH M41
  • 1X TMAC MIX08
  • SAPE M37
  • Wash buffer A MIX12
  • Wash buffer B MIX13
  • 384 well plates M38
  • Nanoscreen tip box cover M30

Assets

  • 7th floor benchtop centrifuge A10
  • Nanoscreen A18
  • Biomek A27
  • Vortex A12
  • Matrix electronic pipette A14
  • 45°C incubator A11
  • 384 well magnets A29

Reagent Mix

ID Name Step Composition Volume/Well Use
MIX06 SAPE master mix SAPE 9.7μl 1X TMAC MIX08
0.3μl SAPE M37
10μl Keep at room temperature and avoid exposure to light

Procedure

TIP: Begin FlexMap 3D scanner warmup for detection step before beginning
SAPE stain in order to save time.
  1. Spin down HYB plates at 3000 rpm for 3 minutes.
    TIP: Check that beads have pelleted in the bottom of plate wells.
  2. Load Biomek program file cmap cleanup that corresponds to the correct number of plates.
  3. Place reagents and plates on deck as follows:INSERT PICTURE
  4. Run Biomek program
    TIP: The program will run the buffer A and B washes, and then pause for the SAPE addition.
  5. Prepare SAPE master mix MIX06
  6. The program will pause. Replace buffer B with reservoir of SAPE master mix and click OK to continue.
    TIP: Program will add 10μl SAPE mix and then pause.
  7. Remove plates from Biomek deck.
  8. Seal HYB plates with MicroSeal A.
  9. Vortex HYB plates to mix beads and SAPE
    IMPORTANT: Ensure that bead pellets are fully dispersed.
  10. Incubate HYB plates in 45°C incubator for 10 minutes.
  11. Spin down plates at 3000RPM for 1 minutes
  12. Remove seals and place HYB plates back on appropriate deck locations.
  13. Select OK to continue running program
  14. Seal plates with MicroSeal foil.
    STOP POINT: If procedure will be continued at a later time, store SAPE plates for up to one week at 4°C in the 7th floor refrigerator A25, sealed tightly with aluminum foil or MicroAmp seals. Be sure to note the date SAPE Stain was completed.
  15. Proceed directly to preparation for Detection (DET) step.

Detection (DET)

Purpose

Read each detection plate with the Luminex FlexMap 3D System, which will determine the identity of each bead based on the internal dye ratio, and the amount of amplicon bound to it by the fluorescence of the SAPE.

IMPORTANT: Prepare materials and assets in a post PCR environment.

Materials

  • Plates from SAPE step

Assets

  • FlexMap 3D scanners A07

Procedure

  1. Prepare FlexMap 3D scanners
    1. Turn on Flexmap 3D Scanners and Computers.
    2. Login to Windows
    3. Login to XPonent software
    4. Eject tray, and fill position RA1 with DDW, RA2 with 70% EtOH
    5. Run maintenance program Daily Instrument Startup
      TIP: Scanner will take 30 minutes to warm up.
  2. Open Batch tab.
  3. Choose New Batch from Existing Protocol.
  4. Select protocol CMAP FINAL.
  5. Eject plate tray.
  6. Load SAPE plate with correct orientation
  7. Run batch.
  8. When complete, run maintenance program Daily Instrument Shutdown.

Probe Pair Preparation

Probe Collapse

Purpose

Collapse probes from 100uM stocks into a single tube.


Preparation

  1. Thaw 100μM probe stocks M54

  2. Label probe aliquot tubes

Materials

  • Cryovials M67

  • Reagent reservoir M16

Assets

  • Water bath A26
  • Vortex A12
  • 20°C freezer, 4th floor A04

Procedure

  1. Label probe aliquot tubes with probe pool version, date collapsed, and initials
  2. Vortex probe plates.
  3. Centrifuge plates for 15 seconds at 1000 RPM to settle liquid in wells.
  4. Uncap probe plates.
  5. Aspirate 2μl from each probe well using a multichannel pipette, and dispense into a reagent reservoir.
    TIP: Change tips each time you aspirate probes to avoid contamination.
  6. Transfer probe collapse set into a Falcon tube.
  7. Vortex collapsed probe set.
  8. Heat probes at 50C in a water bath within a beaker of distilled water for 30 seconds.
  9. Aliquot probe mix in 500μl volumes into Cryovials.
  10. Store probe aliquots in -20°C freezer.

Bead Preparation

Bead Barcode Coupling (BBC)

Purpose

Bind the probe (and therefore target gene) specific LUA tag to each individual bead. To complete a full compiled detection, three detection plates are each coupled to different LUA tags. Calibration genes appear across all three detection plates.

Preparation

  1. Prepare MES buffer MIX14
  2. Thaw LUAs M56

Materials

  • 50 ml Falcon tube M66
  • EDC M58
  • DDW M40
  • MES M62
  • Luminex magnetic microspheres M72
  • Collection microtubes M54
  • 96-well deep well plate M60
  • Reservoir plates M61
  • CyBio 96 well tips M57
  • Reagent reservoir M16
  • Microseal foil M39
  • 96 well magnets A28

Assets

Reagent Mix

ID Name Step Composition Volume/Well Use
MIX14 MES Coupling Buffer BBC 4.88g MES M62
5 drops 5M NaOH M71
249.95ml DDW M40
250ml Bring pH to 4.5, filter sterilize through 0.2μm filter, then store at 4°C

Procedure
IMPORTANT: Complete this process as quickly as possibly to minimize bead exposure to light.

  1. Aliquot 500 ml of Luminex beads in ascending order into racks of 96 deep well plates.
    TIP: Sonicate and vortex Luminex beads thoroughly in their stock bottles before aliquoting. Label plates with correct bead numbers.
  2. Centrifuge plates at 3000 rpm for 1 minute.
  3. Remove buffer from bead plates using CyBio program Remove 500 from 2 DW.
    1. Place 96 well magnets on Position 1 and Position 3 of the CyBio.
    2. Place 96 deep well reservoir in Position 2 to collect waste.
    3. Place a bead coupling plate on each magnet and let sit one minute.
      TIP:The beads will pellet tightly inside the magnetic ring.
    4. Load 96 well CyBio tip cartridge corresponding to the bead set.
    5. Run CyBio program
  4. Add MES using Combi program 65 ul, 96 DW (44 mm) Full Plate.
    1. Bring bead plate to Combi.
    2. Load large cassette into Combi.
    3. Place Combi intake into MES buffer and prime
    4. Run program
  5. Add LUA tags
    1. Thaw LUA plates
    2. Vortex and spin down LUA plates in centrifuge (1000 rpm for 1 min).
    3. Aspirate 2.5 μl of LUA from source plate.
    4. Dispense 2.5 μl of LUA into its designated well in the bead plate, referring to the LUA-Bead layout map.
      TIP:Confirm that LUA is added to each well.
    5. Repeat for all LUA-bead pairs
  6. Add EDC using Combi program 65 ul, 96 DW (44 mm), Full Plate.
    1. Bring all bead plates to Combi.
    2. Load Small Combi cassette.
    3. Mix 50 ml batch of 10 mg/mL EDC in double distilled water (DDW) and pour into reagent reservoir.
    4. Run program for each plate
       IMPORTANT: Complete this process in 5 minutes if possible
    5. Covery with MicroSeal foil
    6. Centrifuge plates for 15 seconds at 1000 RPM to settle liquid in wells.
    7. Incubate in dark at room temperature for 30 minutes.
    8. Repeat EDC addition and incubation for a second time with a new batch of EDC solution. Do not remove EDC from first addition.
  7. Proceed directly to Bead Coupling Wash.

Bead Coupling Wash and Bead Collapse

Purpose

Remove all unbound LUA tags from bead mix to prevent non-specific binding of LUA tags to beads, then collapse beads into sets.

Preparation

  1. Prepare Bead Wash Buffer I MIX10
  2. Prepare Bead Wash Buffer II MIX11

Materials

  • DDW M21
  • Tween-20 M64
  • SDS M65
  • TE buffer M63
  • 96-well deep well plate M60
  • CyBio 96 well tips M57
  • Microseal foil M39
  • Reservoir plates M61
  • 96 well bead magnet A28

Assets

Reagent Mixes

ID Name Step Composition Volume/Well Use
MIX10 Bead Wash Buffer I BBC 50μl Tween-20 M64
249.95ml DDW M40
250ml Filter sterilize through 0.2μm filter, then store at room temperature
MIX11 Bead Wash Buffer II BBC 2.5ml SDS 10% M65
247.5ml DDW M40
250ml Filter sterilize through 0.2μm filter, then store at room temperature

Procedure

  1. Wash beads with Bead Wash Buffer I using CyBio and Combi.
    1. Place 96 well magnets on Position 1 and Position 3 of CyBio.
    2. Place 96 deep well plate in Position 2 of CyBio for waste.
    3. Place a bead coupling plate on top of each 96 well bead magnet and let sit 1 minute.
    4. Run CyBio program Remove 250 from 2 DW.
    5. Bring bead plates to Combi.
    6. Load large cassette into Combi.
    7. Place Combi intake into Bead Wash Buffer I and prime Combi.
    8. Run Combi program 225 ul, 96 DW (44mm), Full Plate.
  2. Wash beads with Bead Wash Buffer II using CyBio and Combi.
    1. Place 96 well magnets on Position 1 and Position 3 of CyBio.
    2. Place 96 deep well plate in Position 2 of CyBio for waste.
    3. Place a bead coupling plate on top of each 96 well bead magnet and let sit 1 minute.
    4. Run CyBio program Remove 250 from 2 DW.
    5. Bring bead plates to Combi.
    6. Load large cassette into Combi.
    7. Place Combi intake into Bead Wash Buffer II and prime Combi.
    8. Run Combi program 225 ul, 96 DW (44mm), Full Plate.
    9. Vortex plate gently to disperse beads
  3. Wash beads with TE Buffer using CyBio and Combi.
    1. Place 96 well magnets on Position 1 and Position 3 of CyBio.
    2. Place 96 deep well plate in Position 2 of CyBio for waste.
    3. Place a bead coupling plate on top of each 96 well bead magnet and let sit 1 minute.
    4. Run CyBio program Remove 250 from 2 DW.
    5. Bring bead plates to Combi.
    6. Load large cassette into Combi.
    7. Place Combi intake into Bead Wash TE and prime Combi.
    8. Run Combi program 225 ul, 96 DW (44mm), Full Plate.
    9. Vortex plate gently to disperse beads
  4. Collapse bead set into a single tube using CyBio.
    1. Place 96 well magnets on Position 1 and Position 3 of CyBio.
    2. Place 96 deep well plate in Position 2 of CyBio for waste.
    3. Place a bead coupling plate on top of each 96 well bead magnet and let sit 1 minute.
    4. Run CyBio program Remove 250 from 2 DW.
    5. Aspirate each well of the bead plate, and collapse into a 50 ml Falcon tube.
    6. Dispense 50 ul of TE into the first column of the aspirated bead plate, then mix and transfer to the next column.
      TIP:Take a second pass through the bead plate to recover as many beads as possible.
    7. Add TE with recovered beads to Falcon tube.

Assets

ID Name Model/Vendor Description Unit/Location
A01 4th floor liquid handler CyBio Vario PERT, prePCR liquid handling, master mix additions CyBio 384, 4th floor
A02 PrePCR thermocycler Thermo MBS Satellite 384 FIRST - LOG, prePCR 4th floor 
A03 PostPCR thermocycler Themro MBS Satellite 384 PCR - HYB, postPCR 7th floor
A04 Freezer, -20°C Sanyo HF-5015 FIRST-LIG, Store LMA reagents CMAP -20°C pre, 4th floor
A05 Freezer −20°C Sanyo MDF-U537 HYB, Store amplified PCR product CMAP -20 post, room 7177
A06 Freezer −80°C Sanyo MDF-U75VC LYSIS, Store cell lysate pre-LMA CMAP -80°C room 4051
A07 FlexMap 3D Luminex DET, Detect LMA product n=9, Room 7177
A08 6 nL slot pin tool V&P Scientific PERT, Add small molecule Pin tool, 4th floor
A09 Benchtop centrifuge Eppendorf 5810 FIRST – LIG, Spin down/out mixes prePCR, 4th floor
A10 Benchtop centrifuge Eppendorf 5810 HYB – WASH, Spin down/out mixes postPCR, room 7177
A11 Incubator 45°C VWR Model 1535 SAPE, Incubate during SAPE stain CMAP incubator, room 7177
A12 Vortex VWR G560 CAP – ANNL, mix liquids CMAP vortex, 4th floor
A13 prePCR multichannel Thermo Matrix 16 Channel CAP - PCR, add mixes to plates CMAP prePCR matrix, 4th floor
A14 postPCR multichannel Thermo Matrix 16 Channel HYB - WASH, add mixes to plates CMAP postPCR matrix, room 7177
A15 4th floor sonicator VWR Ultrasonic, 2L BBC, disperse settled bead CMAP sonicator, 4th foor
A16 Vortex VWR G560 HYB - SAPE, mix liquids CMAP 7th floor vortex, room 7177
A17 7th floor sonicator VWR Ultrasonic, 2L HYB, disperse settled bead CMAP sonicator, room 7177
A18 Liquid handler Nanoscreen HYB, liquid transfer postPCR Nanoscreen 5th floor, Multimek 5th floor
A19 37°C Incubator Thermo Scientific 3110 PLATE, cell culture incubator CMAP TC incubator, 4th floor
A20 Plate rocker Barnstead 4631 LYSIS, plate rocker CMAP plate rocker, 4th floor
A21 CyBio 96 CyBio96 BBC, 96 well liquid transfer CyBio96, 4th floor
A22 Cell counter Beckman Coulter ViCell XR PLATE, cell viability counter ViCell, 4th floor TC room
A23 TC centrifuge Beckman Coulter Allegra X-15R PLATE, tissue culture centrifuge TC centrifuge, 4th floor TC room
A24 TC multichannel Ranin 1200ul pipette PLATE, cell plating multichannel pipette Ranin pipette, 4th floor TC room
A25 4°C fridge SciCool SCGP210W1AREF HYB, storage of post-hyb plates 4°C fridge, room 7177
A26 Water bath Fisher Scientific Isotemp 215 PROBE, prove denaturing Water bath, 4th floor
A27 7th floor liquid handler Biomek NX SAPE - WASH, liquid transfer and wash postPCR Biomek, room 7177
A28 96 well magnets Agencourt BBC, hold down magnetic bead AGN#00219 | 4th floor
A29 384-well magnets Dexter Magnetic WASH, hold down magnetic bead 2501007 | Room 7177
A30 Plate Warmer
A31 Combi
A32 Janus liquid handler
A33 BL2+ centrifuge
A34 Electronic pipette
A35 Aspirating pipette
A36 BL2+ cell culture incubator

Reagents from external vendors

ID Name Vendor Catalog # Process
M01 TCL Lysis Buffer Invitrogen 15596-018 CAP, LYSIS
M02 MicroAmp Seal Applied Biosystems 4306311 CAP - PCR
M03 CyBio 384 tips CyBio OL 3800-25-513-N CAP - PCR
M04 384 well Turbocapture plate Qiagen 72271 CAP - PCR
M05 1.5ml microtubes ISC Bioexpress C-3217-1 CAP
M06 HeLa S3 Total RNA Ambion Am7852 CAP
M07 MCF7 Total RNA Ambion AM7846 CAP
M08 HL60 Total RNA Ambion AM7836 CAP
M09 K562 Total RNA Ambion AM7832 CAP
M10 PC3 Total RNA Ambion AM7834 CAP
M11 MDA-MB-453 Total RNA Ambion AM7876 CAP
M12 Super Rag Mercantile Development 93101 FIRST – LIG
M13 Aluminum Foil Goldman Paper 1217 FIRST – LIG
M14 384-well plate Eppendorf 951020737 FIRST – HYB
M15 15 ml Falcon tubes BD Biosciences 352096 FIRST-STAIN
M16 Reagent Reservoir Corning 4870 FIRST - STAIN
M17 DEPC treated water Ambion AM9915 FIRST
M18 5x M-MLV RT buffer Promega M2505 FIRST
M19 25 mM dNTPs Invitrogen 10297018 FIRST, PCR
M20 M-MLV RT enzyme Promega M2505 FIRST
M21 Nuclease free water Qiagen XXXXX ANNL - PCR
M22 Probe Dilution Plates In-house Custom ANNL
M23 10x Taq ligase buffer NEB M0208L ANNL, LIG
M24 Taq DNA ligase NEB M0208S LIG
M25 10x PCR buffer Qiagen 203443 PCR
M26 Magnesium Chloride Qiagen 203443 PCR
M27 T3 primer IDT - PCR
M28 T7 primer IDT - PCR
M29 HotStarTaq Qiagen 203443 PCR
M30 Nanoscreen tips 30 ul Nanoscreen 30ulnc HYB – WASH
M31 TMAC Sigma T3411-1L HYB, WASH
M32A Coupled Beads In-House Custom HYB
M32B Coupled Beads In-House Custom HYB
M33 20% Sarkosyl Sigma L-9150 HYB
M34 MicroSeal BioRad MSA5001 HYB
M35 1M Tris-HCl, pH 8.0 Sigma T-3038 HYB
M36 0.5M EDTA, pH 8.0 Gibco 15575-038 HYB
M37 SAPE Invitrogen S866 SAPE
M38 384-well plate Costar 3456 SAPE
M39 Microseal F Foil BioRad MSF1001 DET
M40 Distilled Water In House - DET
M41 70% Ethanol In House - DET
M42 384-well cell culture plate Corning 3570 PLATE
M43 PBS MediaTech 21-040-CV PLATE
M44 Trypsin EDTA Invitrogen 25200-114 PLATE
M45 Vi-Cell Sample Vials Beckman Coulter 383721 PLATE
M46 10cm cell culture petri dish BD Falcon 353003 PLATE
M47 RPMI 1640 Media Fisher Scientific MT-10-040-CM PLATE
M48 DMEM Media Invitrogen 11995073 PLATE
M49 DMSO Sigma D2650-100ML PERT
M50 Methanol Fisher A412-4 PERT
M51 1000x CAS compound plate ChemBio Custom PERT
M52 Deep well 384-well plate Costar 3965 LYSIS
M53 Luminex Carboxylated Microspheres Luminex - BBC
M54 Collection Microtubes Qiagen 19560 BBC
M55 100 µM stock probe plates IDT Custom BBC
M56 LUA 96-well stock plates IDT Custom BBC
M57 CyBio 96 well tips CyBio OL 3800-25-559-N BBC
M58 EDC Pierce Biosciences 22980 BBC
M59 96-well plate Axygen 321-21-051 BBC
M60 96-well deep well plate Costar 3960 BBC
M61 Reservoir Plates Axygen Res-SWI-LP BBC
M62 MES Sigma 2933 BBCf
M63 TE Buffer Ambion AM9858 BBC
M64 Tween-20 Boston Bioproducts IBB-180-1L BBC
M65 10% SDS Ambion AM9822 BBC
M66 50mL falcon tubes BD Biosciences 352070 BBC
M67 1mL CryoVials Nunc 377224 PROBE
M68 20X SSPE
M69 12X MES
M70 5M NaCl
M71 5M NaOH
M72 Luminex Magnetic Beads
M73 PCR positive control template
M74 Positive amplicon
M75 50mL conical vials
M76 Combi cassette
M77 Virus
M78 Polybrene media
M79 Puromycin
M80 CTG reagent
M81 MilliQ water
M82 TC plate, RNAi barcoded

Reagents internally prepared

ID Name Step Composition Volume/Well Use
MIX01 MCF7 Total RNA master mix CAP 19.7μl TCL Lysis Buffer M01
0.3μl MCF7 Total RNA M07
20μl Keep on ice, incubate at room temp for 5m before using
MIX02 RT master mix RT 3.7μl DEPC treated water M17
1μl 5X M-MLV RT Buffer M18
0.1μl 25mM dNTPs M19
0.2μl M-MLV RT enzyme M20
5μl Keep on ice, add enzyme when ready to begin master mix addition
MIX03 Probe anneal master mix ANNL 4.22μl Nuclease free water M21
0.28μl "1000" probe pair mix M22
0.5μl 10X Taq ligase buffer M23
5μl Keep at RT, warm probes before adding
MIX04 Ligation master mix LIG 4.4375μl Nuclease free water M21
0.5μl 10X Taq ligase buffer M23
0.0625μl Taq DNA ligase M24
5μl Keep on ice, add enzyme when ready to begin master mix addition
MIX05 PCR master mix PCR 12.768μl Nuclease free water M21
1.5μl 10X PCR buffer M25
0.51μl MgCl2 M24
0.096μl 25mM dNTPs M19
0.015μl T3 primer 100μM M27
0.015μl T7 primer 100μM M28
0.096μl HotStarTaq M29
15μl Keep on ice, add enzyme when ready to begin master mix addition
MIX06 SAPE master mix SAPE 9.7μl 1X TMAC MIX08
0.3μl SAPE M37
10μl Keep at room temperature and avoid exposure to light
MIX07 Coupled bead mix HYB M32A Bead set A (includes controls at 0.5X)
M32B Bead set B (includes controls at 0.5X)
MIX09 1.5X TMAC
21μl Store at 4°C
MIX08 1X TMAC HYB 150ml TMAC M31
1.25ml 20% Sarkosyl M33
12.5ml Tris-HCl M35
2.0ml 0.5M EDTA M36
84.25ml DDW M40
250ml Store at room temperature
MIX09 1.5X TMAC HYB 225ml TMAC M31
1.88ml 20% Sarkosyl M33
18.75ml Tris-HCl M35
3ml 0.5M EDTA M36
1.37ml DDW M40
250ml Store at room temperature
MIX10 Bead Wash Buffer I BBC 50μl Tween-20 M64
249.95ml DDW M40
250ml Filter sterilize through 0.2μm filter, then store at room temperature
MIX11 Bead Wash Buffer II BBC 2.5ml SDS 10% M65
247.5ml DDW M40
250ml Filter sterilize through 0.2μm filter, then store at room temperature
MIX12 Wash Buffer A HYB 300ml 20% SSPE M68
10.2ml 5M NaCl M70
699.9ml DDW M40
1000ml Filter sterilize through 0.2μm filter, then store at room temperature
MIX13 Wash Buffer B HYB 83.3ml 12X MES M69
5.2ml 5M NaCl M70
0.1ml Tween-20 M64
699.9ml DDW M40
1000ml Filter sterilize through 0.2μm filter, then store at room temperature
MIX14 MES Coupling Buffer BBC 4.88g MES M62
5 drops 5M NaOH M71
249.95ml DDW M40
250ml Being pH to 4.5, filter sterilize through 0.2μm filter, then store at 4°C