For general handling instructions refer to the section Materials, the general working recommendations as well as to the respective kit protocols. Storage conditions are described in Table 2. Regarding safety precautions for specific kit components see Table 3. Timing as stated in the following procedure refers to the simultaneous processing of 24 samples.
1. Pre-heat a thermomixer or heated orbital incubator to 56°C. If a thermomixer or heated orbital incubator is not available, a heating block or water bath can be used instead.
2. Using a scalpel, trim excess paraffin off the sample block.
3. Cut one to two sections (5 µm thick) from a histologically confirmed tumor tissue. Troubleshooting. Critical step: if the sample surface has been exposed to air, discard the first 2-3 sections.
4. Immediately place the sections in a 1.5 ml or 2 ml microcentrifuge tube.
Deparaffinization using deparaffinization solution and PK-digestion of FFPE tissue.
Timing: 100 min or overnight.
5. Add 320 µl Deparaffinization Solution (Caution: Irritating) and vortex vigorously for 10 seconds.
6. Add 20 µl distilled water, 15 µl Lysis Buffer FTB, and 5 µl proteinase K.(Caution: Irritating)
Note: A Master Mix comprising distilled water, Lysis Buffer FTB, and proteinase K may be prepared in advance.
7. Vortex and briefly centrifuge to collect the sample in the bottom of the tube.
Note: The Deparaffinization Solution will form a layer above the Lysis Buffer FTB with the addition of proteinase K.
8. Incubate the tubes in a thermal block set to 56°C for 60 min up to overnight to lyse the tissues. Ensure that tissues are completely lysed (the solution will become homogeneous). Pause Point: incubation can also take place overnight.
9. Critical step: Incubate at 90°C ± 5°C for 1 hr ± 5 min.
Note: If using only one heating block, leave the sample at room temperature (15–25°C) after the 56°C incubation, until the heating block has reached 90°C.The incubation at 90°C partially reverses formaldehyde modification of nucleic acids. Longer incubation times or higher incubation temperatures may result in more fragmented DNA. Troubleshooting.
10. Briefly centrifuge the 1.5 ml tube to remove drops from inside the lid.
11. Transfer the complete lysate (about 40 µl, the lower clear phase) in a 200 µl reaction tube or 8-well strip (not provided). Avoid transfer of Deparaffinization Solution as much as possible.
Note: Small amounts of remaining Deparaffinization Solution have no effect on the bisulfite reaction.
Bisulfite conversion of lysate and bisDNA (bisulfite converted DNA) cleanup
Timing: 232 min.
Make sure the Bisulfite Solution (Caution: irritant) is completely dissolved. Critical step: do not place dissolved Bisulfite Solution on ice.
12. Prepare the bisulfite reactions in 200 µl PCR tubes (not provided). Add each component in the order listed in Table 5.
Critical step: when using the therascreen PITX2 RGQ PCR Kit, the “Low concentration” protocol from the EpiTect Fast Bisulfite Conversion Handbook must always be used, as the concentration of gDNA purified from FFPE samples is usually low.
Critical step: the bisulfite mix should be immediately vortexed for 5 sec after adding the DNA Protect Buffer (caution: reproductive toxicity, irritant) to protect samples from degradation. Troubleshooting.
13. Close the PCR tubes and mix immediately the bisulfite reactions thoroughly. Store the tubes at room temperature (15–25°C).
Critical step: DNA Protect Buffer should turn from green to blue after addition to the DNA-Bisulfite Solution mixture, indicating sufficient mixing and correct pH for the bisulfite conversion reaction, or DNA binding to the MinElute DNA spin column. Troubleshooting.
14. Perform the bisulfite DNA conversion using a thermal cycler. Program the thermal cycler according to Table 6. The complete cycle should take ~30 min. If using a thermal cycler that does not allow to enter the reaction volume (140 µl), set the instrument to the largest volume setting available.
15. Place the PCR tubes containing the bisulfite reactions into the thermal cycler. Start the thermal cycling incubation.
Critical step: as the bisulfite reaction is not overlaid with mineral oil, only thermal cyclers with heated lids are suitable for this procedure. It is important to use PCR tubes that close tightly.
Pause Point: converted DNA can be left in the thermal cycler overnight (at 20°C) without any loss of qPCR assay performance.
16. Briefly centrifuge the PCR tubes containing the bisulfite reactions. Then transfer the complete bisulfite reactions to clean 1.5 ml microcentrifuge tubes.
17. Add 310 µl Buffer BL (Caution: harmful) to each sample. Mix the solution by vortexing and then centrifuge briefly.
18. Add 250 µl ethanol (96–100 %) to each sample. Mix the solutions by pulse vortexing for 15 sec and centrifuge briefly to remove the drops from inside the lid.
19. Transfer the entire mixture from each tube into the corresponding MinElute DNA spin column.
20. Centrifuge the spin columns at maximum speed for 1 min. Discard the flow-through and place the spin columns back into the collection tubes.
21. Add 500 µl Buffer BW (wash buffer) to each spin column, and centrifuge at maximum speed for 1 min. Discard the flow-through and place the spin columns back into the collection tubes.
22. Add 500 µl Buffer BD (desulfonation buffer) to each spin column and incubate for 15 min at room temperature (15–25°C). (Caution: corrosive) If there are precipitates in Buffer BD, avoid transferring them to the spin columns.
Critical step: the bottle containing Buffer BD should be closed immediately after use to avoid acidification from carbon dioxide in the air.
Note: it is important to close the lids of the spin columns before incubation. Troubleshooting.
23. Centrifuge the spin columns at maximum speed for 1 min. Discard the flow-through and place the spin columns back into the collection tubes.
24. Add 500 µl Buffer BW to each spin column and centrifuge at maximum speed for 1 min. Discard the flow-through and place the spin columns back into the collection tubes.
25. Add 500 µl Buffer BW a second time to each spin column and centrifuge at maximum speed for 1 min. Discard the flow-through and place the spin columns back into the collection tube.
26. Add 250 µl ethanol (96–100%) to each spin column and centrifuge at maximum speed for 1 min.
27. Place the spin columns into new 2 ml collection tubes and centrifuge the spin columns at maximum speed for 1 min to remove any residual liquid.
28. Place the spin columns with open lids into a clean 1.5 ml microcentrifuge tube (not provided) and incubate the columns for 5 min at 60°C in a heating block.
Critical step: this step ensures the evaporation of any remaining liquid.
29. Add 15 µl Buffer EB (elution buffer) directly onto the center of each spin-column membrane and close the lids gently.
Critical step: do not elute with less than 15 µl buffer as the eluate volume would be too small to proceed with the qPCR step.
30. Incubate the spin columns at room temperature for 1 min.
31. Centrifuge for 1 min at 15,000 x g (12,000 rpm) to elute the DNA.
Pause point: we recommend storing purified DNA at 2–8°C for up to 24 hrs. When storing purified DNA for longer than 24 hrs, we recommend storage at –30 to –15°C.
DNA concentration determination - Timing: 25 min.
32. The concentration of DNA is determined by measuring absorbance at 260 nm following the instrument procedure using QIAGEN’s QIAxpert for example (ssDNA plugin: single stranded nucleic acid measurement) or a NanoDrop instrument. Absorbance readings at 260 nm should fall between 0.1 and 1.0 to be accurate. An absorbance of 1 unit at 260 nm corresponds to 33 µg of bisDNA per ml (A260 = 1 = 33 µg/ml). Note: if using the QIAxpert ssDNA plug-in, an EB-blank sample has to be included, which is after measurement automatically subtracted from the OD values. Ideally, the minimal gDNA concentration is 6 ng/µl but samples as low as 2 ng/µl may be processed with risk of “Low input” invalid results. Pause point: bisDNA samples can be stored at -20°C until further use.
qPCR set up, run and analysis - Timing: 105 min.
The therascreen PITX2 RGQ PCR Kit must be run on the Rotor-Gene Q MDx 5plex HRM instrument using automated interpretation of results with Rotor-Gene AssayManager v2.1. Take time to familiarize yourself with the Rotor-Gene Q MDx instrument and with the Rotor-Gene AssayManager v2.1 software before starting the protocol. See the respective user manuals for the instrument, Rotor-Gene AssayManager v2.1, and the Gamma Plug-in for details.
33. Cool a Loading Block 72 x 0.1 ml tubes for 10 min in a deep-freezer or for at least 1 hr at refrigerator temperature.
34. Thaw all therascreen PITX2 RGQ PCR Kit components and samples in a refrigerator, on ice, on a cooling block or at room temperature for as long as necessary.
35. Place the thawed products on ice, on a cooling block or in the refrigerator until placing them back into –30 to –15°C after use.
Note: therascreen PITX2 RGQ PCR Kit components can be kept at 2–8°C and protected from light for a maximum of 6 hr if used several times the same day.
36. Vortex the tubes (10–12 sec), then centrifuge them briefly before use. Except PITX2 RGQ PCR MMx, which is mixed by pipetting up and down as it contains Taq Polymerase.
37. Prepare PITX2 qPCR reaction mix on ice (or using a cooling block) in a 1.5 ml or 2 ml tube according to the number of samples to be processed.
The pipetting scheme for the preparation of the PITX2 reaction mix, shown in Table 7, is calculated to achieve final reaction volumes of 20 µl after addition of 4 µl bisDNA sample or control. Extra volume is included to compensate for pipetting errors and to allow preparation of sufficient reaction mix for four samples tested in duplicate, plus four controls. If less samples are tested, the reaction mix can be prepared accordingly. Remember to allow for the extra volume to compensate for pipetting errors (one extra well for up to 10 wells and two extra wells for up to 20 wells). Troubleshooting.
38. Vortex (10–12 sec) and briefly centrifuge the PITX2 qPCR reaction mix. Place the qPCR strip tubes on a pre-cooled Loading Block 72 and dispense 16 µl of the PITX2 qPCR reaction mix per strip tube following the example of loading block setup shown in Figure 2.
The numbers denote positions in the loading block and indicate final rotor position. The positions of the controls are set in the PITX2 assay profile and cannot be changed. If controls are not placed as indicated, the automated result analysis cannot be performed. REF50: PITX2 RGQ PCR Reference 50; REFlow: PITX2 RGQ PCR Reference Low; NC: PITX2 RGQ PCR Negative Control, NTC: PITX2 RGQ PCR NTC (NTC); Sample 1 to 4: bisDNA samples, NA: empty well. Troubleshooting.
39. Vortex (10–12 seconds) and briefly centrifuge bisDNA samples, PITX2 RGQ PCR Reference 50 (Ref50), PITX2 RGQ PCR Reference Low (REFlow), PITX2 RGQ PCR Negative Control (NC) and PITX2 RGQ PCR NTC (NTC). Troubleshooting.
40. Add 4 µl sample or control material into its corresponding tube according to the setup in Figure 2 to obtain a total volume of 20 µl. Mix gently 5 times by pipetting up and down. Note: be careful to change tips between each tube to avoid false-positive results from contamination by any nonspecific template. Troubleshooting.
41. Close all tubes and check that no bubbles are present at the bottom of the tubes. Troubleshooting.
42. Return all the therascreen PITX2 RGQ PCR Kit components and samples to the appropriate storage conditions to avoid any material degradation. Pause point: it is highly recommended to start the run as soon as possible after the preparation, however, if the tubes are prepared but cannot be processed directly (e.g. due to instrument unavailability), it is possible to store the plate at 2–8°C and protected from light up to 24 hrs. Troubleshooting.
43. Place a 72-Well Rotor on the Rotor-Gene Q MDx rotor holder.
44. Fill the rotor with strip tubes previously prepared according to the assigned positions, starting at position 1. Critical step: make sure the first tube is inserted into position 1 and the strip tubes are placed in the correct orientation and positions (important for run validity and traceability of sample). Always keep the four controls (REF50, REFlow, NC and NTC) in positions 1 to 4 so that gain optimization (performed on tube position 1) is always performed on the same control sample. Make sure controls are loaded in the correct order for the automated analysis of the controls (an inversion of controls will invalidate the run by the PITX2 assay profile). Troubleshooting.
45. Fill empty positions with empty, closed tubes to fill the rotor entirely.
46. Attach the locking ring.
47. Load the Rotor-Gene Q MDx instrument with the rotor and locking ring. Close the instrument lid.
Creating a work list and starting the qPCR run
48. Switch on the Rotor-Gene Q MDx instrument.
49. Open the Rotor-Gene AssayManager software by clicking the icon: The Rotor-Gene AssayManager window opens (Figure 3).
50. Log in as a user with the “Operator” role in the closed mode. Click “OK“. The Rotor-Gene AssayManager screen opens (Figure 4).
51. Check that the RGQ is correctly detected to the software before launching the run.
52. Select the “Setup” tab. Note: The overall functionalities of the Setup environment and of “Creating/Editing a Work List” are described in the Rotor-Gene AssayManager v2.1 Core Application User Manual.
53. Click “New work list” (Figure 4).
54. Select the PITX2 assay profile from the list of available assay profiles (Figure 5).
55. Transfer the selected assay profile to the list of selected assay profiles by clicking on the arrow (to the right of the assay profile name). The assay profile should now be displayed in the selected assay profiles list (Figure 5).
56. In the “Assays” tab, complete the yellow fields: number of samples in accordance with your plate setup (Figure 6). Note: the number of samples does not correspond to the number of wells and does not include controls. Samples are tested in duplicates; therefore, one sample corresponds to two wells. For example, the number of samples to be inserted is 4 for the plate of 12 wells presented in Figure 2.
57. Select the “Kit Information” tab. Insert the kit information by either selecting “Use kit bar code” (and scan the bar code) or selecting “Enter kit information manually” and inserting manually the kit information found on the label of the therascreen PITX2 RGQ PCR Kit box:
1) Material number
2) Expiry date
3) Lot number
58. Select the “Samples” tab. A list with the sample details is shown. This list represents the expected layout of the rotor.
59. Enter the sample identification as well as any optional sample information as a comment for each sample (Figure 7).
60. Select “Properties” and enter a work list name (Figure 8; “a”).
61. Enable the check box “worklist is complete (can be applied)”( Figure 8; “b”).
62. Save the work list (Figure 8; “c”). Optional: press “Print work list” to print the work list. Printing the work list may help with the preparation and setup of the run. The sample details are included as part of the work list.
63. Select the corresponding work list from the work list manager and click “Apply” (Figure 8; “d”). Alternatively, if the work list is still open, click “Apply”. Note: check that the Rotor-Gene Q MDx is correctly detected by the software before launching the run.
64. Enter the experiment name.
65. Select the cycler to be used in “Cycler Selection”.
66. Check that the locking ring is correctly attached and confirm on the screen that the locking ring is attached.
67. Click “Start run”. The qPCR run should start. Troubleshooting.
Release and report qPCR results
The general functionality of the approval environment is described in the Rotor-Gene AssayManager v2.1 Gamma Plug-in User Manual. After a run has been finished and the cycler has been released, the experiment will be saved in the internal database. The analysis of the acquired data is performed automatically according to the rules and parameter values defined by the assay profile.
68. When the run has finished, click on “Finish run” to analyze and export data. Critical step: until this step is completed, the experiment is not saved in the internal database.
69. After clicking “Finish run”, enter the password and click “Release and go to approval” (Figure 9).
· For users logged in with the “Approver” role, click “Release and go to approval”.
· For users logged in with the “Operator” role, click “Release”.
· If “Release and go to approval” was clicked, the results for the experiment are displayed in the “Approval” environment.
· If “Release” was clicked by a user with the “Operator” role, someone with an “Approver” role must log in and select the “Approval” environment. Note: in the “Approval” tab, experiments can be analyzed by shifting between each tab (i.e., experiment, assay, audit, trail, run control results).
70. Check the amplification curves for each sample, tick the first box on the right side of the “flags” column (the box becomes green) (Figure 10). Troubleshooting.
71. Click “Release/report data” (at the bottom right of the window) to create a .pdf report and to save the LIMS file. A copy is automatically saved in the following location:
72. Close the pdf file and return to the Rotor-Gene AssayManager. Click “OK” each time it is asked.
73. Go to the “Archive” tab to export the .rex file (Figure 11; “a”). Check that “start date” and “end date” are correct (Figure 11; “b”) and click “apply filter”. (Figure 11; “c”) Select the experiment to export (Figure 11; “d”) then click on “Show assays” (Figure 11; “e”).
74. Export the.rex file (the file is saved in C:\Documents and settings\AllUsers\Documents \QIAGEN\RotorGeneAssayManager\Export\Experiments).
Note: the software automatically generated a LIMS file in the following location:
C:\Documents and settings\All Users\Documents\QIAGEN\RotorGeneAssayManager\Export\LIMS
75. Unload the Rotor-Gene Q MDx instrument and discard the strip tubes according to your local safety regulations.