The BioVision Retrovirus qPCR Quantitation Kit is used for the quantification of retroviruses. This kit employs a rapid RNA extraction step that can be conveniently and directly coupled with a qRT-PCR reaction. It is a simple one-step titration assay designed to offer high sensitivity and specificity. In this kit, the threshold cycle (Ct) values of the viral lysate and the retroviral standards provided are determined. by qRT-PCR. The Ct values are then used to calculate the retroviral titer of the virus samples.
- An ideal tool to quantify retroviruses
- Reliable and ready to use
- Results ready in less than 2 h
- High specificity and sensitivity
- Non-specific minimum fund
- Virus-producing cell lines or purified viral preparations.
User-supplied reagents and equipment:
- 2X qPCR dye-based master mix
Note: select the appropriate master mix for your specific instrument. See BioVision’s Jade ™ Master Mix catalogue. Nos. (M1105- M1108) that come in a variety of formulations. Each Master Mix has been optimized for performance according to the QPCR machine and reference dye (Mix (No dye) = no reference dye (ROX) dye, bicycle = for bicycle, low ROX = for low ROX, ROX = regular ROX).
- qPCR Thermal Cycler
- PCR tubes
- 1X phosphate-buffered saline
Shipping and storage conditions:
The kit should be stored at -20 ° C upon arrival. Avoid repeated freeze-thaw cycles. All reagents are stable for up to 12 months when properly stored at -20 ° C.
Virus samples originating from both virus-producing cell lines and purified viral preparations can be used to determine the titer. The virus lysis buffer included in the kit helps to extract the viral RNA from the virus sample (the extracted RNA is called “viral lysate”). The Reagent Mix and 2X qPCR MasterMix Retrovirus (not included) are used with the viral lysate and retroviral standards. (STD1 / STD2) to determine the threshold cycle (Ct) values by qRT-PCR. The resulting Ct values are then used to calculate the retroviral titer of virus samples.
1. Sample preparation:
For purified viral samples: dilute the virus to the range of 106 -108 pfu/ml with 1X phosphate-buffered saline before subjecting the virus to viral lysis. For viral samples directly from a virus-producing cell line: collect the culture medium and Centrifuge for 5 min at 2000 g to remove cells/debris. The resulting supernatant will undergo viral lysis. Please wash the cells after transfection and change the medium before cell harvesting. This is to help remove plasmid DNA that could interfere with the titer. results.
2. Viral lysis:
Add 2 μl of the diluted purified viral preparation or supernatant (from step 1) to 18 μl of Viral Lysis Buffer and incubate at room temperature for 3 min. This solution is now called a viral lysate. The Ct value of the viral lysate will be used to determine the titer of the unknown viral sample. Note: note that in this step the viral sample has been diluted 1:10; therefore the dilution factor must be taken into account when calculating the titer.
3. qRT-PCR setup:
Set up reactions as shown below with 2X qPCR Mastermix (not included) in this kit.
4. Recommendations for optimal results:
1. Start the titer essay as soon as the virus is produced or thawed.
2. Upon receipt, aliquot all components to minimize freeze-thaw cycles and reagent contamination.
3. Always keep reaction components and mixtures on the ice during setup. Initiate qPCR as soon as the reaction mixtures have been prepared.