Standard Operating Procedure (SOP)

Title: Fragment Analyzer – RNA (Eukaryotic) run using method DNF-471-33 – SS Total RNA 15nt

SOP ID: FA-RNA-DNF471-33

Version: 1.0 Effective Date: 2025-11-07 Prepared by: — Approved by: —

1. Purpose

Run total RNA on the Fragment Analyzer using the eukaryotic RNA analysis mode with the method DNF-471-33 – SS Total RNA 15nt. This SOP encodes the run summary parameters provided and standardizes execution, QC, and recordkeeping.

2. Scope

Applicable to laboratory staff operating the Fragment Analyzer for eukaryotic RNA quality assessment using the DNF-471-33 method.

3. Responsibilities

• Operator: Perform checks, prepare consumables, execute run, verify QC, archive data.
• Supervisor: Ensure operator training and periodic review of QC trends.

4. Safety and PPE

• Wear lab coat, safety glasses, and nitrile gloves.
• Handle gels, buffers, and cleaning solutions according to SDS.
• Use waste containers for gel/buffer disposal per local regulations.
• Avoid electrical contact with electrodes; de-energize before maintenance.

5. Materials and Equipment

• Fragment Analyzer instrument, Device Serial #: 4172.

• Capillary array: 33 cm effective length; 7 capillaries installed.

  • Array Serial #: 100418-06SFS; log Array Usage Count each run.

• Software: FA Version 1.2.0.11 (or later, validated).

• Consumables per DNF-471-33 kit: gel, buffer(s), ladder/marker as specified by supplier.

• 96‑well plate(s) for samples/standards.

• RNase-free tips, tubes, and plates.

• Optional: RNase inhibitor-treated water for dilutions.

6. Definitions

• Full Conditioning: Manufacturer-defined array conditioning prior to use.
• Gel Prime to Buffer: Prime gel toward buffer reservoir as configured.
• Electrode Rinse Dips: Number of immersion cycles in rinse well.

7. Pre‑Run Checks

1. Confirm instrument status and calibration is current.
2. Inspect capillary array for wear; ensure usage is below manufacturer limit; record Array Usage Count prior to run.
3. Verify sufficient gel and buffer volumes; check waste bottle.
4. Confirm analysis method is present and unmodified: DNF-471-33 – SS Total RNA 15nt.
5. Confirm Analysis Mode: RNA (Eukaryotic).
6. Confirm tray layout matches this SOP.
7. Ensure samples are free of debris; spin down plates briefly; keep samples on ice until loading.
8. Create run folder and file naming per Section 11.

8. Plate and Tray Layout

• Tray Name: Tray-1.
• Load sample plate(s) in the configured tray position(s).
• Place rinse well(s) as defined below in Section 10 (Electrode Rinse Settings).
• Load gel and buffer per kit instructions.
• Load ladder/marker per kit instructions. Note: the provided run summary indicates Marker 1: No; if a ladder is required by kit, load as specified and update records accordingly.

9. Method Parameters (Programmed Settings)

Use the following exact settings:

Note: Two “Rinse” entries exist in the source summary. Implement no global rinse step, but perform electrode rinse: Tray 3, Row A, 2 dips.

10. Procedure

10.1 Instrument and Software Setup

1. Power on the instrument. Launch the FA software (v1.2.0.11 or validated newer).
2. Connect to Device Serial 4172. Confirm array details and usage count.
3. Select method DNF-471-33 – SS Total RNA 15nt. Confirm parameters match Section 9.

10.2 Consumable Loading

1. Load Gel 1 into the gel position per manufacturer instructions.
2. Load running buffer into the buffer reservoir.
3. If a ladder/marker is required by the kit, load into designated well(s).
4. Prepare rinse well in Tray 3, Row A for 2 dips.

10.3 Plate Loading

1. Aliquot RNA samples into a 96‑well plate using RNase‑free technique.
2. Seal plate to prevent evaporation.
3. Place plate in Tray‑1 in the configured slot.
4. Enter sample identifiers in the software sample table.

10.4 Conditioning and Pre‑Run

1. Run Full Conditioning: Yes.
2. Run Gel Prime to Buffer: Yes.
3. Ensure Gel Prime: No (do not run separate gel prime).
4. Perform Prerun at 8.0 kV for 30 s.

10.5 Rinse

• Global Rinse: No.
• Electrode Rinse: Execute 2 dips in Tray 3, Row A as configured.

10.6 Sample Injection and Separation

1. Sample Injection: 5.0 kV for 4 s.
2. Separation: 8.0 kV for 40.0 min.
3. Monitor current and pressure traces; abort if abnormal values persist.

10.7 Analysis

1. Verify Analysis Mode: RNA (Eukaryotic).
2. Review electropherograms and virtual gel.
3. If software provides RIN/RQN or equivalent, record values for each sample.

11. Data and File Management

• Run file name and path (example from source): C:\AATI\Data\2022 05 17\15-50-46\2022 05 17 15H 50M.raw
• Adopt this structure: C:\AATI\Data\YYYY MM DD\HH-mm-ss\YYYY MM DD HH'H' mm'M'.raw
• Save run summary and export CSV/PDF results to the same folder.
• Backup data to the lab server within 24 h.
• Record the following in the run log: date/time, operator, device serial, array serial, array usage pre/post, method name, gel lot, buffer lot, any deviations, QC outcomes.

12. QC and Acceptance Criteria

• Baseline: Stable, no persistent spikes.
• Ladder/Marker: Loaded per kit (if used). Peaks resolve as expected.
• Negative control: No unexpected peaks.
• Replicates: Technical replicates show comparable peak profiles.
• Array health: Usage within manufacturer limits; replace if excessive tailing or poor resolution.
• Run validity: Current and voltage within expected ranges; separation completes without faults.
• Data integrity: Files saved to defined path; checksums optional.

13. Troubleshooting

• No peaks: Check injection voltage/time, sample concentration, electrode contact.
• Broad peaks/poor resolution: Inspect gel age, array health, full conditioning status, and temperature.
• High noise: Replace buffers, check for bubbles, verify proper gel loading.
• Carryover: Increase electrode rinse dips; insert a blank between samples.
• Aborted run: Document fault code; repeat after resolving root cause.

14. Records

Maintain a bound or electronic log with: operator, date/time, SOP ID/version, device and array serials, usage count, consumable lots, method parameters (Section 9), acceptance outcomes (Section 12), and data path.

15. References

• Kit/method instructions for DNF-471-33 – SS Total RNA 15nt.
• Instrument user manual and software guide.
• Laboratory QA manual.