Ionic G2 FFPE Complete Kit

By Bionano Genomics, Inc.

G2 Ionic FFPE Complete Protocol NOTE: Use one Ionic® FFPE to Pure RNA Kit and one Ionic® FFPE to Pure DNA Kit G2 Ionic FFPE Complete Kit Protocol, CG-00039, Rev. C For Research Use Only. Not for use in diagnostic procedures. Revision History REVISION NOTES A Initial release B Changed amount of Proteinase K added during the Preparation of Lysates section to 135 ul. C Complete using G2 FFPE to DNA Kit and G2 FFPE to RNA Kit NOTE: This protocol now requires the use of two kits: One Ionic® FFPE to Pure RNA Kit One Ionic® FFPE to Pure DNA Kit Kit Contents Table 1: Contents provided in one Ionic® FFPE to Pure RNA Kit and one Ionic® FFPE to Pure DNA Kit Container Item Name Description Volume Quantity per Kit -20⁰C Reagents Box Lysis Buffer 1 Lysis Buffer 1 1.35 mL 12 -20⁰C Reagents Box Proteinase K Proteinase K Reagent 810 μL 2 -20⁰C Reagents Box DNase I DNase I Reagent 120 μL 1 -20⁰C Reagents Box RNase RNase A Reagent 120 μL 1 -20⁰C Reagents Box Sample Buffer Blank Sample Buffer 1.6 mL 2 RT Reagents Box Lysis Buffer 2 Lysis Buffer 2 3.8 mL 2 RT Reagents Box 1 - Extraction Buffer Extraction Buffer 12 mL 2 RT Reagents Box 2 - Anodic Buffer Anodic Buffer 12 mL 2 RT Reagents Box 3 - Separation Buffer Separation Buffer 18 mL 2 RT Reagents Box 4 - Neutralization Buffer Neutralization Buffer 12 mL 2 RT Reagents Box 5 - Cathodic Buffer Cathodic Buffer 12 mL 2 RT Reagents Box Mineral Oil Mineral Oil 15 mL 2 Fluidic Chip Set Box Ionic Fluidic Chip Fluidic Chips N/A 12 Table 2: Reagents provided by the user. User-supplied Reagents For Lysate Preparation For Purification FFPE sections or scrolls Table 3: Equipment provided by the user. User-supplied Equipment (or equivalent) For Lysate Preparation For Purification Razors/scalpels (if using slides) 12-column reservoir (Agilent 204365-100) P200 multichannel pipette P200 single channel pipette P20 single channel pipette Microcentrifuge Programmable ThermoMixer Vortex mixer (adjustable speed) Table 4: Labware provided by the user. User-supplied Labware (or equivalent) For Lysate Preparation For Purification DNA LoBind Tube, 1.5 mL (Eppendorf 22431021) Optional: DNA LoBind Tube, 2.0 mL (Eppendorf 22431048) Optional: DNA LoBind Plate, 96-well (Eppendorf 951032000) Protocol at a Glance Lysate Preparation 1. Centrifuge FFPE tissue sample tubes for 2 minutes 2. Add 300 μL of mineral oil 3. Prepare Lysis Mix 1 and add 325 μL to each sample 4. Incubate samples on ThermoMixer. • 65°C for 5 mins at 1000 rpm • 60°C for 1 hour at 500 rpm • 70°C for 1 hour at 0 rpm 5. Transfer 155 μL of lysate for RNA processing into a separate tube then proceed with RNA purification 6. Incubate remaining lysate for DNA processing containing mineral oil on Thermomixer then proceed to DNA purification. • 70°C for 7 hours at 1000 rpm • Hold at 8°C RNA Purification 1. Prepare RNA Lysis Mix 2 and add 65 μL to each tube for RNA purification. 2. Incubate on ThermoMixer • 20°C for 10 mins at 300 rpm 3. Prepare Purification Buffer Reservoir 3. Place Ionic Fluidic Chip onto instrument 4. Load Purification Buffers and prime Ionic Fluidic Chip 6. Add 200 μL of each lysate to the chip 7. Start purification run (~70 minutes) 8. Collect ~50 μL of each extract DNA Purification 1. Centrifuge tubes for DNA processing for 5 minutes and transfer lysates to new tubes 2. Prepare DNA Lysis Mix 2 and add 65 μL to each tube 3. Incubate on ThermoMixer • 20°C for 10 mins at 300 rpm 4. Prepare Purification Buffer Reservoir 5. Place Ionic Fluidic Chip onto instrument 6. Load Purification Buffers and prime Ionic Fluidic Chip 7. Add 200 μL of each lysate to the chip 8. Start purification run (~70 minutes) 9. Collect ~50 μL of each extract Sample Requirements This protocol is intended for the independent extraction and purification of RNA and DNA from up to eight Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples in parallel and is optimized to recover the maximum amount of RNA and DNA from a minimal amount of tissue. Bionano recommends a single 10 μm section of FFPE tissue (scroll or slide-mounted) with an area of 50–300 mm 2 for most downstream applications. For this protocol, a 10 μm section of FFPE tissue with dimensions of 1 inch x 1 inch (25.4 mm x 25.4 mm) and a mass of < 12 mg is typical. The optimal amount of starting material will be informed by the FFPE tissue thickness, cross-sectional area, and cellularity. If a 10 μm section yields <1 μg of RNA or DNA with traditional kits (bead- or column-based), increasing the input FFPE tissue amount for RNA purification on the Ionic Purification System is acceptable. In such cases, this protocol can accommodate FFPE tissue sections totaling 20 μm in thickness (e.g., 2 x 10 μm or 4 x 5 μm).Care must be taken to avoid overloading since adding too much input FFPE tissue can lead to lower than expected RNA or DNA recovery (see Figure 1 ). A small pilot study is helpful in defining optimal input conditions as some FFPE tissue samples present challenges to extraction and purification requiring optimization beyond the standard protocol. Contact support@bionano.com for guidance on processing samples that do not meet the above requirements. Figure 1. Impact of overloading Lysis Procedure Overview of the Approach The Ionic FFPE Complete Kit provides DNA and RNA from the same starting FFPE sample by splitting the sample after lysis into two tubes, digesting the non-target nucleic acid, and then purifying the lysates on the Ionic Purification System. A summary of this procedure is shown in Figure 2. Figure 2. Overview of purification approach and pause points for the FFPE Complete Kit Preparation of Lysates 1. Transfer each FFPE tissue section into a 1.5 mL LoBind Eppendorf tube. 2. Centrifuge tubes containing FFPE tissue sections at maximum speed (>10,000x g) for 2 minutes to move tissue to the bottom of the tube. Hold at room temperature. 3. Place two tubes of Lysis Buffer 1 on the ThermoMixer and incubate at 56°C for 10 minutes at 1000 rpm. If a precipitate is present after incubation, place tube back on ThermoMixer until buffer is clear. 4. Vortex the Lysis Buffer 1 tubes for 3 seconds, pulse spin and immediately proceed to the next step. Hold at room temperature. 5. Remove Proteinase K from the freezer, flick three times and pulse spin. Hold on ice. 6. To prepare Lysis Mix 1, add 135 μL of Proteinase K directly to each Lysis Buffer 1 tube. Vortex for 3 seconds and pulse spin. Hold at room temperature. 7. Add 300 μL of mineral oil to each sample tube from Step 2. 8. Using a P200 pipette, add 325 μL of prepared Lysis Mix 1 to each sample tube with two 162.5 μL transfers. Change tips between each dispense. NOTE: If a precipitate is present in Lysis Mix 1, heat at 56°C until the solution is clear before adding to tubes. Do not vortex sample tube containing mineral oil and Lysis Mix 1. 9. Place sample tubes into a ThermoMixer and incubate using a program with the following steps: Incubate at 65°C for 5 minutes at 1000 rpm. Incubate at 60°C for 1 hour at 500 rpm. Incubate at 70°C for 1 hour at 0 rpm. 10. After the program is completed, remove tubes from the ThermoMixer, pulse spin, and hold on the benchtop for 5 minutes. Set ThermoMixer to 20°C in preparation of the DNase treatment below. NOTE: The mixture will separate into two phases with the nucleic acid-containing lysate in the lower phase. 11. With a P200 pipette set to 155 μL, gently pipette mix the lower nucleic acid -containing lysate 10 times. 12. Aspirate 155 μL of lysate and transfer to a new tube for RNA processing. Hold these RNA lysates on ice. NOTE: To preserve RNA integrity, proceed immediately to DNase treatment. Alternatively, store lysate tubes at -20°C for up to 7 days. Frozen lysates must be thawed on ice prior to proceeding to DNase treatment. 13. Hold tubes containing mineral oil and the remaining lysate from Step 11 at room temperature for DNA processing. Proceed with the Processing DNA Lysates section below immediately after the RNA lysates have completed the DNase treatment and the ThermoMixer is available. DNase Treatment of RNA Lysates 1. Remove DNase I from the freezer, flick three times and pulse spin. Hold on ice. 2. To prepare ‘RNA Lysis Mix 2’, combine 630 μL of Lysis Buffer 2 and 20 μL of DNase I in a 1.5 mL tube. Invert ‘Lysis Mix 2’ ten times to mix and pulse spin. Hold on ice. 3. Add 65 μL of ‘RNA Lysis Mix 2’ to each lysate tube for RNA processing from Step 12 of the Preparation of Lysates section above while on ice. 4. Invert lysate tubes ten times, pulse spin and incubate on a ThermoMixer at 20°C for 10 minutes at 300 rpm. 5. Vortex lysate tubes for 10 full seconds and pulse spin. Hold on ice for at least 5 minutes and proceed directly to Purification. Processing DNA Lysates 1. Place lysates for DNA processing from Step 13 of the Preparation of Lysates section above into a ThermoMixer and incubate using a program with the following steps: Incubate at 70°C for 7 hours at 1000 rpm. Hold at 8°C. 2. After the program is completed, remove tubes from ThermoMixer and hold on the benchtop for 5 minutes. Set ThermoMixer to 20°C in preparation of the RNase treatment below. NOTE: If mineral oil layer is solidified, place at 20°C or higher until mineral oil is no longer solid. 3. Centrifuge tubes at maximum speed (>10,000x g) for 5 minutes. NOTE: The mixture will separate into two phases with the nucleic acid-containing lysate in the lower phase. If mineral oil layer is solidified, place at 20°C or higher until mineral oil is no longer solid. 4. Using a P200 pipette with the tip touching the bottom of the tube, slowly aspirate and transfer 155 μL of lysate from the lower phase of each tube into new microtubes avoiding any pelleted material that may be present. Hold lysate tubes on ice. NOTE: A minimal amount (~5-10 μL ) of mineral oil may be aspirated during transfer of the lysate and does not impact the purification process. To preserve DNA integrity, proceed immediately to the next step. Alternatively, store lysate tubes at -20°C for up to 7 days. Frozen lysates should be thawed on ice prior to proceeding to RNase treatment. RNase Treatment of DNA Lysates 1. Remove RNase from the freezer, flick three times and pulse spin. Hold on ice. 2. To prepare DNA Lysis Mix 2, combine 630 μL of Lysis Buffer 2 and 20 μL of RNase in a 1.5 mL tube. Invert Lysis Mix 2 ten times to mix and pulse spin. Hold on ice. 3. Add 65 μL of DNA Lysis Mix 2 to each lysate tube for DNA processing from Step 4 of the Processing DNA Lysates section above while on ice. 4. Invert lysate tubes ten times, pulse spin and incubate on a ThermoMixer at 20°C for 10 minutes at 300 rpm. 5. Vortex lysate tubes for 10 full seconds and pulse spin. Hold on ice for at least 5 minutes and proceed directly to Purification. Purification Prepare Buffer Reservoir 1. Label a 12-channel reservoir as shown in Figure 2 skipping every other column to prevent purification buffer cross-contamination. Figure 2. Reservoir column labels 2. Add purification buffers to each column according to Table 5. Table 5. Reservoir buffers and volumes Reservoir Column Buffer from Kit Volume 11 - Extraction Buffer 2.0 mL 22 - Anodic Buffer 2.0 mL 33 - Separation Buffer 3.0 mL 44 - Neutralization Buffer 2.0 mL 55 - Cathodic Buffer 2.0 mL Set Up Ionic Purification Run 1. From the Ionic Purification System start screen, press Start then select a User profile for the run. 2. Press the G2 FFPE Complete button. The instrument cover will open. 3. When prompted, remove a fluidic chip from its packaging, handling only by the side skirting, and place it on the instrument stage with the barcode on the right as shown in Figure 3. Gently apply pressure to all four corners of the fluidic chip simultaneously to confirm that the chip is fully seated on the stage. Press the Arrow on the right side of the screen to continue. NOTE: Remove fluidic chip from its packaging and place chip directly on the instrument stage to minimize accumulation of static electricity. Avoid contact with the top and bottom surfaces of the fluidic chip. Figure 3. Proper handling and placement orientation of an Ionic fluidic chip. 4. Enter Run Name, Chip ID, and Reagent Lot and press the Arrow on the right side of the screen to c