0 item(s)
View cart
You have no items in your shopping cart.
Product Description
SHENTEK® Reverse Transcriptase Assay Kit is a research-use kit for detecting reverse transcriptase (RT) activity using MS2 RNA as template, followed by reverse transcription and fluorescent qPCR detection. The assay follows the reverse transcriptase activity test method referenced in the Chinese Pharmacopoeia. The kit is designed for testing animal cell matrices used during production and verification of biological products. Note: certain cell types (e.g., chicken embryonic fibroblasts, other avian-origin cells, and some rodent-derived cell lines) may show endogenous RT activity and can test positive; users should follow applicable regulatory requirements when interpreting results.
Technical Specifications
| Parameter | Details |
|---|---|
| Intended use | Detection of reverse transcriptase activity in cell matrices and related samples using MS2 RNA template, reverse transcription, and fluorescent qPCR |
| Kit format / reactions | Reagents for 50 reactions |
| Validated sensitivity / LOD | Validated that all 10 replicate RT samples of ST8 (10^4 pU/μL) are detected (10/10) |
| Standard curve range (typical) | Typical recommended standard points ST3–ST8 corresponding to a 10-fold serial dilution series ending at ST8 = 10^4 pU/μL (implied range approximately 10^9 to 10^4 pU/μL depending on ST0 preparation) |
| Quantitation acceptance criteria | Standard curve R² ≥ 0.96; Positive control sample (PCS) Ct ≤ 28; sensitivity sample Ct ≤ 38 |
| Result interpretation thresholds | Negative: no Ct or Ct ≥ 40 without amplification curve. Positive: Ct < 40 with clear amplification curve. Borderline: Ct between 38–40 -> repeat test; if repeated Ct < 40 with clear curve then positive. |
| RT incubation | 37 °C for 4 hours (to produce reverse transcription product) |
| qPCR reaction volume | 30 μL total volume per qPCR well (25 μL qPCR mix + 5 μL RT product) |
| RT reaction volume | 25 μL total per RT reaction (20 μL RT-MIX + 5 μL sample/standard) |
| qPCR cycling program (example, ABI 7500 SDS v1.4) | RNase digestion 37 °C 07:00 (1 cycle); Activation 95 °C 05:00 (1 cycle); 50 cycles: Denature 95 °C 00:20, Anneal/Read 57 °C 01:00 (fluorescence read), Extend 72 °C 00:10; Final extend 72 °C 02:00 (1 cycle) |
| qPCR reagents composition (per reaction) | 2×qPCR SHENmix 15 μL; MS2 Primer & Probe MIX 3 μL; RNase A (10 mg/mL, user-supplied) 1 μL; 100×ROX 0–0.3 μL (instrument dependent); ddH2O to 25 μL |
| RNase handling | qPCR Reaction MIX contains RNase A (user-supplied addition). Prepare/spike RNase A separate from reverse transcription workspace to avoid contamination. |
Features
- MS2 RNA template-based assay: Uses MS2 RNA as an external template for sensitive detection of reverse transcriptase activity.
- Reverse transcription followed by fluorescent qPCR: Assay couples reverse transcription with fluorescent qPCR for quantitative detection and standard curve absolute quantitation.
- Complies with referenced pharmacopoeia method: Protocol is aligned with the reverse transcriptase activity test method described in the Chinese Pharmacopoeia.
- Pre-formulated controls and mixes: Includes M-MLVRT control, MS2 Primer & Probe mix, and 2×qPCR SHENmix to simplify assay setup.
- Validated sensitivity procedure: Includes recommended sensitivity validation (10 replicates of lowest standard ST8) to confirm assay performance on first use.
- Designed for cell matrix testing: Intended for screening reverse transcriptase activity in animal cell matrices used in biological product development and verification.
Applications
- Detection of reverse transcriptase activity in cell culture supernatants or cell matrix preparations
- Screening of cell substrates for retroviral reverse transcriptase activity during biological product production and release testing
- Verification testing of biological raw materials and process intermediates for RT contamination
- Research applications investigating retroviral-like activity in samples
Kit Contents
M-MLVRT Control (NNA024), 6 μL × 1 tube-20 °C
ddH2O (NND010), 1 mL × 1 tube2–8 °C
Buffer A (NND012), 1.5 mL × 2 tubes2–8 °C
Buffer B (NND013), 750 μL × 1 tube2–8 °C
MS2 RNA (NND011), 15 μL × 1 tube-20 °C
Reverse Transcription Buffer (NNB010), 1 mL × 1 tube-20 °C, protect from light
MS2 Primer & Probe MIX (NNC036), 150 μL × 1 tube2–8 °C (or per label)
2×qPCR SHENmix (NNC045), 1 mL × 1 tube2–8 °C
100×ROX (NND007), 20 μL × 1 tube2–8 °C, protect from light
* Total: 50 reactions | Method: Reverse transcription followed by fluorescent qPCR (absolute quantitation using standard curve)
Attention
• For Research Use Only (RUO). Not for diagnostic use.
• Read Material Safety Data Sheets (MSDS) and follow handling instructions.
• Wear appropriate PPE (eyewear, mask, lab coat, gloves).
• Use RNase-free consumables and work in RNase-controlled environment; UV-irradiate surfaces and equipment and disinfect with 75% ethanol as recommended.
• Prepare and spike RNase A into qPCR mix in an area separate from reverse transcription operations to avoid contamination.
• Protect light-sensitive reagents (e.g., ROX, reverse transcription buffer) from light and store as indicated.
• Positive controls should be aliquoted and stored at −65 °C; contact manufacturer to order PC if needed.
• Certain cell types (e.g., chicken embryonic fibroblasts, avian-origin cells, and some rodent cell lines) may contain endogenous retroviral sequences and commonly test positive; interpret according to regulatory guidance.
• If test sample Ct is between 38–40, repeat the test. Confirm positive only if repeat Ct < 40 with clear amplification curve.
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: How should I store the kit and its components?
Store frozen components (e.g., M-MLVRT Control, MS2 RNA, Reverse Transcription Buffer) at −20 °C. Other reagents such as primers/probe mix and 2×qPCR SHENmix should be stored per label (typically 2–8 °C). Protect light-sensitive reagents (e.g., ROX, RT buffer) from light. The kit is stable for up to 12 months under appropriate storage.
Q2: What is the validated detection sensitivity of the assay?
The kit validation procedure requires that all 10 replicate RT samples of ST8 (10^4 pU/μL) are detected (10/10). ST8 is used as the sensitivity check; users should perform the sensitivity test on first use.
Q3: How are results interpreted (positive/negative)?
Negative: no Ct or Ct ≥ 40 without an amplification curve. Positive: Ct < 40 with a clear amplification curve. If Ct is between 38–40, repeat the test; if the repeat Ct < 40 with a clear curve, consider the sample positive.
Q4: What acceptance criteria must be met for a valid run?
Standard curve R² ≥ 0.96; Positive control (PCS) Ct ≤ 28; sensitivity sample Ct ≤ 38. If these criteria are not met, the run should be considered invalid and repeated.
Research Use Only
✔
Research Use Only (RUO). Method aligned with reverse transcriptase activity test method referenced in the Chinese Pharmacopoeia. – 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.
