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For Research Use Only (RUO)
$1,751.00
Sku: 1201201-1
Product Description
SHENTEK® Residual E. coli RNA Quantitation Kit (2G) is designed for quantitation of host cell RNA from Escherichia coli in biopharmaceutical products. The kit employs a duplex reverse transcription quantitative PCR (qRT-PCR) format including a target detector (FAM) and an Internal Positive Control (IPC, VIC) in the E. coli RNA Primer & Probe MIX. The assay provides rapid, specific and reliable quantitation with femtogram-level sensitivity for the target gene. The kit includes One Step qPCR Buffer, One Step Enzyme MIX, Primer & Probe MIX (incl. IPC), E. coli RNA Control and RNase-free water, and is compatible with common real-time PCR instruments.
Technical Specifications
| Parameter | Details |
|---|---|
| Detection chemistry | Duplex one-step reverse transcription quantitative PCR (one-step RT-qPCR) with FAM-labeled target and VIC-labeled IPC |
| Sensitivity / Limit of detection | Target detectable at femtogram (fg) level (document states target gene can be determined at the femtogram level) |
| Standard curve concentration range | Serial standards prepared from 2000 pg/µL (stock) producing working standards: 20, 2, 0.2, 0.02, 0.002 pg/µL (ST1–ST5) |
| Reaction volume | Total reaction volume: 20 µL (15 µL qRT-PCR MIX + 5 µL sample/standard/controls) |
| qRT-PCR mix composition per well | One Step qPCR Buffer 10 µL, One Step Enzyme MIX 1 µL, E. coli RNA Primer & Probe MIX (incl. IPC) 4 µL — total 15 µL |
| Thermal cycling program | Reverse transcription 50 °C 15:00 (1 cycle); Activation 95 °C 0:30 (1 cycle); 45 cycles of 95 °C 0:10 (denature) and 60 °C 0:40 (anneal/extend, fluorescence read) |
| Analysis settings (recommended) | Manual Ct; Threshold 0.05; Automatic baseline; ROX as passive reference (for ABI 7500), set Standard Curve (Absolute Quantitation) |
| Controls and acceptance criteria | NTC Ct should be at least 2 cycles higher than the lowest standard; ERC recovery acceptance 50%–150%; compare sample IPC Ct to NTC/NCS IPC Ct to evaluate inhibition |
| DNase digestion conditions (for plasmid samples) | Incubate samples with DNase I at 37 °C for 30–60 minutes; recommended DNase I final concentration 0.2–2 U/µL depending on sample; ensure plasmid sample ≤100 ng/µL |
| DNase inactivation | Options: (1) SHENTEK® Residual Host Cell RNA Sample Preparation Kit purification; (2) heat inactivation at 75 °C for 10 minutes; (3) other validated methods |
| Storage & stability (kit) | Store kit components at –20 °C; kit components stable up to 24 months (check label expiration) |
Features
- Duplex one‑step RT-qPCR: Combined reverse transcription and qPCR in a single reaction for speed and reduced handling
- High sensitivity: Target detection at femtogram (fg) level enabling quantitation of trace residual E. coli RNA
- Internal Positive Control (IPC): IPC included in Primer & Probe MIX (VIC) to monitor PCR performance and potential inhibition
- Ready-to-use reagents: Pre-formulated buffer, enzyme mix and primer/probe mix to simplify setup and improve reproducibility
- Instrument compatibility: Validated workflow compatible with common real-time PCR systems (e.g., SHENTEK-96S, ABI 7500, LightCycler 480 II, Bio-Rad CFX96)
- Validated workflow for biopharmaceutical samples: Protocols and optional sample prep kit provided for plasmid DNA and protein expression products
Applications
- Quantitation of residual host cell RNA (HCR) from Escherichia coli in biopharmaceutical products
- Residual RNA testing in plasmid DNA preparations
- Residual RNA testing in protein expression products derived from E. coli
- Method validation and in-process or release testing for downstream product quality control
Kit Contents
E. coli RNA Control (NNA011) 50 µL × 1 tube–20 °C
One Step qPCR Buffer (NNB008) 500 µL × 2 tubes–20 °C, protect from light
One Step Enzyme MIX (NNC052) 100 µL × 1 tube–20 °C, protect from light
E. coli RNA Primer & Probe MIX (incl. IPC) (NNC119) 400 µL × 1 tube–20 °C, protect from light
RNase-Free H2O (NND008) 1.2 mL × 3 tubes–20 °C
* Total: Reagents for 100 Reactions | Method: qRT-PCR (duplex one-step reverse transcription quantitative PCR)
Attention
• For Research Use Only (RUO). Not for diagnostic or therapeutic use.
• Read Material Safety Data Sheets (MSDS) and follow handling instructions; wear appropriate PPE (eyewear, mask, gloves, lab coat).
• Protect light-sensitive components (buffer and enzyme mix) from light; store all kit components at –20 °C and check expiration date.
• Thaw reagents completely at 2–8 °C or on ice and mix gently; avoid repeated freeze-thaw cycles.
• Work in RNase-free conditions: use RNase/DNase-free consumables, decontaminate surfaces and irradiate equipment with UV for 30 minutes, disinfect with 75% ethanol.
• Perform DNase I treatment to remove genomic DNA for plasmid-derived samples; incubate at 37 °C for 30–60 minutes and inactivate DNase I (e.g., 75 °C for 10 minutes or purification).
• Ensure final plasmid concentration ≤100 ng/µL when performing DNase digestion; use DNase I final concentration 0.2–2 U/µL as appropriate.
• Include controls: standards (ST1–ST5), NTC, NCS and ERC; ERC recovery should be within 50%–150% to accept sample results.
• If sample IPC Ct is significantly higher than NTC/NCS IPC Ct, sample inhibition is possible; interpret IPC as reference and consider sample recovery.
Quality Management & Certifications
Quality System
Download QMSQMS (ISO, GMP)
Download CertificateQuality Advantages
View ReportQuality Control Process
Download ProcessTechnical Resources & Downloads
Frequently Asked Questions (FAQ)
Q1: What is the analytical sensitivity (limit of detection) of the kit?
The kit is reported to detect the target gene at the femtogram (fg) level. Use the provided standard curve (typical working standards: 20 to 0.002 pg/µL) to determine assay sensitivity in your system and matrix.
Q2: Which instruments are compatible with this kit?
The kit is compatible with common real-time PCR instruments including SHENTEK-96S, Applied Biosystems 7500, Roche LightCycler 480 II and Bio-Rad CFX96. Program settings may require adjustment per instrument/software.
Q3: How should samples derived from plasmid DNA be prepared?
Plasmid-derived samples require DNase I digestion to remove genomic DNA (37 °C for 30–60 min). After digestion, inactivate or remove DNase I (e.g., heat 75 °C for 10 min or use the SHENTEK Residual Host Cell RNA Sample Preparation Kit) before qRT-PCR.
Q4: What controls should be included and what are acceptance criteria?
Include a standard curve (ST1–ST5), no template control (NTC), negative control sample (NCS), and extraction recovery control (ERC). NTC Ct should be ≥2 cycles above the lowest standard; ERC recovery should be between 50% and 150%.
Research Use Only
✔
Research Use Only (RUO) – This product is intended for laboratory research purposes only and not for clinical or regulatory diagnostic use.
✔
Not intended for use in USDA or FDA regulated diagnostic testing or official compliance testing.
When can I expect my order to ship?
Most orders are filled and shipped within 2-3 business days from the time they are received.
Our standard shipping usually take 2-5 days.
We also provide express shippping for time-sensitive deliveries.
Email contact@biofargo.com if you have any requirements.
