Dear Renas,

we also use extracted RNA from vaccines as positive controls in our PCRs for example. For that, we resuspend the lyophilized live vaccines in virus transport medium rather than water (DEPC or RNAse/DNase free water) to preserve virus viability and RNA integrity longer.

As you say, the vaccine vials contains a dose for a high number of animals, also meaning that the virus concentration is high. Depending on your application, you may need to adjust this concentration, f.i. to decrease the chance of cross-contaminations when handling high virus loads.

You can resuspend your vaccine vial f.i. in 25 ml of virus transport medium. For that you can use 2-5 ml to resuspend the lyophylisat directly in the bottle, transfer it to a 50ml falcon tube, then rince the vaccine vial with another 2-5 ml of medium to rince the vial, transfer to the falcon tube. Then adjust the volume in your falcon tube to 25 ml and vortex the tube to resuspend and homogenize properly.

When it is homogeneous, you can certainly aliquot your solution and store it at -80°C for future use, to create a reference stock etc.

No problem for using commercial kits to extract RNA. We have been extracted RNA from vaccine using QIAmp viral RNA minikit from Qiagen without problems.

For virus transport medium (VTM) receipe, you can check Snoeck et al. 2011 J Gen Virol. But there are other receipe for VTM. F.i. adding BSA in virus preservation medium can really help preserving their viability.

Hope this helps

Chantal

Hello, see attached info may be useful for you.

Good luck!

Hi

You should add some distilled water by syringe inside the vaccine vial. then shake it well. Under laminar flow hood, extract 0.5 cc of suspension and used it for RNA extraction.

Regards

Mohammad Malekan thanks alot for the answer, but how much water should I add?

Your welcome

It depends on the size of your vaccine vial. normally 1or 2 cc could be enough.

By the way it is better to use Diethyl pyrocarbonate (DEPC) water instead of distilled water for vaccine preparation.

Renas Isa,

To prepare the vaccine for RNA extraction, you need to add DEPC water to dissolve the 'solidified' vaccine as mentioned by Mohammad Malekan . You should however know that, the type of vaccine in the vial may considerably affect the success of your experiment.

I want to assume you will PCR amplify some portion of the vaccine after RNA extraction. If the vaccine is based on whole virus ( as in the case of live attenuated or inactivated vaccine), you may not face any challenges. However, if the vaccine is a subunit vaccine or a recombinant vectored vaccine in which only some portion of the virus is included and that portion happens to be not what you are interested in amplifying, you may end up having some difficulties. Therefore, try accertaining the type of vaccine you are dealing with.

I wish you good luck in your research

Bashir

I agree with Bello

Dear Renas,

we also use extracted RNA from vaccines as positive controls in our PCRs for example. For that, we resuspend the lyophilized live vaccines in virus transport medium rather than water (DEPC or RNAse/DNase free water) to preserve virus viability and RNA integrity longer.

As you say, the vaccine vials contains a dose for a high number of animals, also meaning that the virus concentration is high. Depending on your application, you may need to adjust this concentration, f.i. to decrease the chance of cross-contaminations when handling high virus loads.

You can resuspend your vaccine vial f.i. in 25 ml of virus transport medium. For that you can use 2-5 ml to resuspend the lyophylisat directly in the bottle, transfer it to a 50ml falcon tube, then rince the vaccine vial with another 2-5 ml of medium to rince the vial, transfer to the falcon tube. Then adjust the volume in your falcon tube to 25 ml and vortex the tube to resuspend and homogenize properly.

When it is homogeneous, you can certainly aliquot your solution and store it at -80°C for future use, to create a reference stock etc.

No problem for using commercial kits to extract RNA. We have been extracted RNA from vaccine using QIAmp viral RNA minikit from Qiagen without problems.

For virus transport medium (VTM) receipe, you can check Snoeck et al. 2011 J Gen Virol. But there are other receipe for VTM. F.i. adding BSA in virus preservation medium can really help preserving their viability.

Hope this helps

Chantal

Very professional, extensive, and helpful reply by Chantal! I concur the suggestion to dilute the stock if it is going to be used as a control for PCR reactions or extract separately from the samples of interest.

If you are going do perform RT-qPCR for IBV, another tip is to calibrate your positive control (diluted vaccine) for Cq value of +- 25,00, otherwise, if you use so much virus you may easily cross-contaminate your samples. For this purpose, you may run a ten-fold serial dilutions, extract the RNAs and run the RT-qPCR. The dilution that gives a Cq around 25 may be selected for positive control.

Resuspend the lyophilized live vaccines in virus transport medium (DEPC or RNAse/DNase free water but don't use water for you to kept the virus in a good condition by preserving virus viability and maintain RNA integrity.

All the best.

many thanks for your precious answers. These answers saved my life thanks again