Ultra-Sensitive Detection : Detects residual K562 feeder cells at levels as low as 0.05% (10 copies/μL), ensuring compliance with FDA guidelines for cell therapy safety.
RT-PCR-Based Specificity : Utilizes optimized primers/probes targeting K562-specific sequences via RT-PCR, validated for specificity, accuracy, and precision per regulatory requirements.
Wide Dynamic Range : Linear detection range of 10–1×10<sup>6</sup> copies/μL, covering trace residuals to high-concentration samples without dilution.
Ready-to-Use & Streamlined Workflow : Includes pre-diluted quantitative standards and a 2.5-hour total assay time (RNA-to-results), reducing hands-on steps and variability.
Regulatory-Compliant Performance : Validated with ~100% recovery, CV% ≤ 5%, and R² ≥ 0.990 amplification efficiency, meeting CDE/FDA standards for process characterization.
Broad Instrument Compatibility : Compatible with major qPCR platforms (e.g., ABI 7500, Bio-Rad CFX96, Roche LightCycler 480) and includes ROX reference dyes for flexible deployment.
Kit Components: Thaw all reagents (except primers/probes) at 4°C, then vortex and briefly centrifuge (6000 rpm, 5 sec).
Safety: Wear PPE (lab coat, gloves, mask) and work in a clean area to avoid contamination.
Equipment Setup:
Ensure qPCR instrument (e.g., ABI 7500, Bio-Rad CFX96) is calibrated with FAM/VIC channels.
Prepare RNA extraction and reverse transcription reagents.
RNA Extraction:
Isolate total RNA from 1×10<sup>6</sup>–5×10<sup>6</sup> cells using a commercial RNA extraction kit.
Reverse Transcription:
Convert RNA to cDNA using gDNA removal and RT reagents (not included in kit).
Standard Curve Dilution:
Serially dilute the K562 Quantitative Standard (1×10<sup>8</sup> copies/μL) with DNA Diluent to generate 6 points (10–1×10<sup>6</sup> copies/μL).
Example:
STD | Dilution Ratio | Final Concentration |
STD1 | 10μL STD0 + 90μL Diluent | 1×10<sup>6</sup> copies/μL |
... | ... | ... |
STD6 | 10μL STD5 + 90μL Diluent | 10 copies/μL |
Master Mix Assembly (per reaction):
Component | Volume (μL) |
2× qPCR Buffer | 10 |
Primer/Probe Mix | 4.6 |
50× ROX* | 0.4 |
*Omit ROX if instrument does not require it (e.g., Roche LightCycler).
Plate Setup:
Load 15μL Master Mix + 5μL sample/standard per well (triplicates for standards/samples). Include NTC (DNA Diluent).
| Milestones | Specifications | Deliverables |
| Kit Design & Optimization | • Target-specific primer/probe design for K562 feeder cell detection via RT-PCR. | • Optimized primer/probe sequences. |
| • Validation for specificity, accuracy, and precision per FDA/CDE guidelines. | • Validation report (LOD: 0.05%, linearity: 10–1×10<sup>6</sup> copies/μL). | |
| Ready-to-Use Components | • Pre-diluted quantitative standards (1×10<sup>8</sup> copies/μL). | • 100-test kit with standardized reagents (-18°C stable for 18 months). |
| • Pre-mixed 2× qPCR buffer and ROX reference dyes. | • Reduced hands-on time (no dilution steps). | |
| Rapid Workflow | • Total assay time: 2.5 hours (RNA extraction to results). | • Streamlined protocol with step-by-step guidance. |
| • Compatible with major qPCR platforms (ABI 7500, Bio-Rad CFX96, Roche LightCycler). | • Broad instrument flexibility (ROX High/Low options). | |
| Ultra-Sensitive Detection | • Detection limit: 10 copies/μL (0.05% residual K562 cells). | • Reliable trace-level analysis for cell therapy QC. |
| • Dynamic range: 10–1×10<sup>6</sup> copies/μL. | • FDA-aligned sensitivity for feeder cell safety. | |
| QC & Performance | • CV% ≤ 5% (replicates), R² ≥ 0.990, 85–110% amplification efficiency. | • SDS-PAGE/Western Blot validation data (optional). |
| • Includes NTC and extraction recovery controls. | • Excel-based data analysis template. | |
| Regulatory Support | • Compliant with FDA guidelines for residual feeder cell detection in cell/gene therapies. | • Documentation for regulatory submissions. |
| • CDE-approved validation parameters. | • Research-use-only disclaimer. |
Purpose: Ensure sensitive and accurate detection of residual K562 feeder cells in cellular therapy products.
Features:
Utilizes RT-PCR with optimized primers/probes targeting K562-specific sequences.
Detection range: 10–1×10<sup>6</sup> copies/μL, with a 0.05% sensitivity limit (FDA-compliant).
Includes pre-diluted standards and qPCR master mix for streamlined workflow.
Applications:
Quality control for NK cell expansion and other K562-based co-culture systems.
Validated for specificity, precision (CV% ≤ 5%), and linearity (R² ≥ 0.990).
Purpose: Minimize hands-on time while maintaining high reproducibility.
Features:
Ready-to-use reagents: Eliminate dilution steps (e.g., pre-mixed 2× qPCR buffer, ROX dyes).
Total assay time: 2.5 hours (RNA extraction to results).
Compatible with major qPCR instruments (e.g., ABI 7500, Bio-Rad CFX96, Roche LightCycler).
Technical Support:
Includes Excel-based data analysis template for automated residual % calculation.
Step-by-step protocol with NTC and extraction controls to ensure reliability.
Purpose: Support compliance with FDA/CDE guidelines for cell therapy safety.
Features:
Pre-validated parameters: Amplification efficiency (85–110%), recovery (~100%), and LOD verification.
Documentation: Detailed reports for regulatory submissions (e.g., specificity, precision data).
Stability: Reagents stable at -18°C for 18 months (freeze-thaw tested).
Applications:
Critical for GMP-compliant manufacturing of cell/gene therapies.
Research-use-only disclaimer aligned with ethical standards.
Background:
A biotech company developing NK cell therapies used irradiated K562 feeder cells to enhance expansion efficiency (10,000-fold increase). FDA guidelines required rigorous residual detection.
Challenge:
Needed sensitivity ≤0.05% to meet safety thresholds.
Existing methods (flow cytometry) lacked precision for trace-level detection.
Solution:
Deployed the K562 RT-PCR Detection Kit with:
Pre-optimized primers/probes for K562-specific targets.
10–1×10<sup>6</sup> copies/μL dynamic range.
Validated per FDA/CDE guidelines (CV% ≤5%, R² ≥0.990).
Result:
Detected 0.03% residual K562 cells (below FDA-recommended threshold).
2.5-hour workflow accelerated batch release vs. 8 hours with ddPCR.
Approved for Phase II trials with full regulatory documentation.
Background:
A CAR-T manufacturer used K562 feeders for T-cell activation but faced challenges quantifying residuals in final products.
Challenge:
Required GMP-compliant, high-throughput testing.
Cross-reactivity with patient-derived cells skewed earlier results.
Solution:
Implemented the kit’s FAM/VIC duplex assay (K562 vs. ABL control).
Ready-to-use standards eliminated dilution errors.
Integrated with Bio-Rad CFX384 for 96-well scalability.
Result:
Achieved 99% specificity (no false positives in patient samples).
Recovery rate ~100% across spiked samples.
Reduced QC costs by 40% vs. outsourced testing.
Background:
An academic lab studied NK cell-mediated clearance of irradiated K562 cells but lacked sensitive tools to track residuals over time.
Challenge:
Needed quantitative data at multiple time points (0–72 hours).
Traditional microscopy was low-throughput and subjective.
Solution:
Used the kit’s RT-PCR workflow to measure K562 DNA degradation:
RNA-to-cDNA conversion optimized for degraded samples.
Excel template automated % residual calculations.
Result:
Identified complete clearance by 48 hours (0.01% detection limit).
Published data in Cell Therapy Journal with kit methodology cited.
1-805-304-6109 
info@hillgene.com 
English
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