Principle and Procedure
RNA viruses are lysed by Lysis Buffer VLB1. Addition of Carrier RNA improves binding and recovery of low concentrated viral RNA. The RNA binds to the membrane and contaminants (potential PCR inhibitors) like salts, metabolites and soluble macromolecular cellular
components are removed efficiently in two wash steps using two different buffers VWB and VWB3. The nucleic acids can be eluted in low salt buffer or water (RNase free) and are ready-foruse in subsequent downstream applications like PCR, real time PCR etc. The high quality purified RNA is free of any contaminants and inhibitors; and ready for direct use or safe storage.
Specifications of RNASure Virus Kits:
RNASure Virus kits are designed for the rapid preparation of highly pure viral nucleic acids (e.g.
HCV, HIV, DENV, H1N1) from fluid biological samples e.g. serum, plasma, body fluid, cell
cultured supernatant, and from transport medium of swabs. RNASure Virus kit is intended for
general laboratory use. RNASure Virus kit is suitable for 150μL of sample as starting material.
Yield & quality of the prepared nucleic acid is suitable for further application like DNA
Sequencing, RT-PCR, RNA dot blots, cDNA transcription, Taqman analysis and array
technologies. The detection limit of certain viruses depends upon the individual procedures. We recommend
using internal (low copy) standards as well as positive and negative control to monitor the
purification, amplification and detection processes. The procedure is designed to avoid Sample
to Sample cross contaminations and allow safe handling of infectious samples.
Starting Material and Typical Yields
Upto 50 µg of RNA can be extracted from upto 150 µl Serum, Plasma, Cell-free biological fluid in 50 µl elution volume using RNASure Virus Kit. Viral Nucleic Acid Isolation [ 5]
Handling Samples
Viral nucleic acid can be purified from various sources e.g., serum, plasma, urine and from transport medium of nasopharyngeal swabs. Samples may be fresh or frozen, frozen samples
should not be thawed more than once. Incubation with buffer VLB1 can be prolonged to dissolve and digest residual cell structures, precipitates and virus particle. Viral RNA is sensitive to
autolysis and prolonged incubation may decrease the yields.
Preparation and Storage of Reagents
Precaution:
Please wear gloves & safety goggles. All buffers & kit components stored at room temperature (18-25 C) are stable for at least 12 months.
Carrier RNA
Add 1ml of lysis buffer VLB1 to the complete content of the carrier RNA tube, dissolve & transfer it
back to the VLB1 bottle. Lysis buffer VLB1 including carrier RNA must be stored at 4ºC. Aliquot and store at -20 C for longer periods. Storage at 4 C may cause salt precipitation, preheat at 40 -60 C for 5 minutes to re-dissolve salts.
Note: Do not warm Buffer VLB1 containing Carrier RNA more than 4 times.
Buffer VWB and VWB3
Reconstitute Buffer VWB by adding 8 ml of ethanol (96-100%). Similarly add 80 ml of ethanol (96- 100%) to reconstitute Buffer VWB3 before use. Buffer VWB and VWB3 can be stored at room temperature (18-25ºC).