QIAamp® Viral RNA Mini Kit From Qiagen

The QIAamp® Viral RNA Mini Kit is used for isolation of viral RNA which can then be utilized in amplification technologies, for example, reverse transcriptase PCR (RT-PCR). All buffers and reagents provided with the kit are guaranteed to be ribonuclease-free. Also provided are RNase-free 2 ml collection tubes and Mini Spin columns, which can be used with a microcentrifuge or a vacuum manifold. Qiagen’s QIAamp® Viral RNA Mini Kit does not require any unpleasant phenol/chloroform extractions or ethanol precipitations.

Typical problems with RNA extraction include degradation due to omnipresent RNases and contamination with DNA. When using this kit, there will be cellular DNA contamination if it is present in the sample. One can circumvent this issue either by utilizing cell-free body fluids or simply by prior centrifugation of the sample and using only the supernatant for RNA extraction. Another possibility is to treat the sample with DNase, which is unfortunately not included in the kit. I used this kit for viral RNA isolation from cell culture supernatants and respiratory samples such as pharyngeal aspirates and sputum. All extractions have been carried out successfully.

Before use, the lysis buffer (AVL) has to be supplemented with carrier RNA (provided). It is very important to check this buffer prior to use for precipitates, otherwise you will have very low yields of viral RNA in most cases, as lysis cannot take place properly. During the lysis step, all potential infectious particles are inactivated.

Qiagen states successful isolation of viral RNA from HIV, HAV, HCV, HDV and enteroviruses. I worked with a variety of coronaviruses, all of which delivered good RNA yields of good purity and integrity. However, due to supplementation of carrier RNA to the lysis buffer, the amount of viral RNA is difficult to determine photometrically, as most of the RNA present is carrier RNA (5.6 ug per 140 ul sample).

The whole protocol from lysis to elution only takes 20 to 30 minutes. Several samples can be handled simultaneously. Normally, 140 ul plasma, serum, urine or cell culture supernatant is used per purification. But it is also possible to scale the sample volumes up to 560 ul if necessary. Moreover, one can concentrate body fluid samples where only low titers are suspected (but you will need microconcentrators in this case; they are not provided with the kit).

To summarize, I have had only good experiences using this kit for the isolation of coronaviral RNA from cell culture supernatants and patient samples. Purification is straightforward and fast. Although I did not determine RNA yields after elution due to the carrier RNA, all RT-PCR experiments which were conducted using the eluted viral RNA as template were successful.