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. 2022 Jun 9;12(1):70.
doi: 10.1186/s13568-022-01413-x.

In-house protocol: spin-based viral RNA purification

Mahmoud M Abdelfattah  1 , Ahmed M Osman  2 , Mohamed A Elnagar  3 , Mohamed F Ibrahim  3 , Magdy Albert  3 , Maya M Talal  3 , Nasra F Abdel Fattah  2 , Samah A Loutfy  2 , Reham Helwa  4
Affiliations

In-house protocol: spin-based viral RNA purification

Mahmoud M Abdelfattah et al. AMB Express. .

Abstract

A worldwide shortage of molecular biology consumables is in surge. This includes filter tips, nucleic acid purification kits, polymerases, reverse-transcriptase, and different types of reagents which are included in viral diagnostic kits. In developing countries, the problem is even worse, since there is few capital enterprise to adopt this kind of industry. So, our aim is to develop a suitable, functional, comparable to commercial ones, and affordable in-house protocol to purify viral RNA. We sought some published and commercial RNA purification solutions to set-up an in-house protocol for viral RNA extraction. Solution was prepared accordingly. Also, LPA (linearized polyacrylamide) carrier was evaluated. The whole setting of in-house solutions with addition of LPA carrier was compared to QIAamp viral RNA minikit solutions. Our results showed that linearized polyacrylamide (LPA) carrier in homemade solutions is comparable to poly A carrier which is used in the most commercial kit. In addition, the whole setting of RNA purification solutions did achieve the purpose of viral RNA purification. Also, the result was confirmed using sputum of a Sars-Cov2 infected patient. Our experiments did end up with an affordable homemade solutions for viral RNA purification.

Keywords: In-house protocol; LPA carrier; RNA purification; Vero cell line; Viral RNA.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1
Fig. 1
Experiment flow chart of viral RNA purification. A kit solutions. B procedure summary
Fig. 2
Fig. 2
The validation workflow of the in-house protocol
Fig. 3
Fig. 3
Evaluation of LPA carrier versus Qiagen carrier. RNA was purified from infected Vero cells with measles virus. The same amount of cells used as startup point to compare Qiagen carrier and LPA carrier using all Qiagen reagents (i.e. the only difference between the tubes is type of carrier). A RT-PCR of N-450 using MeV210 and MeV217 primers for RNA purified with Qiagen reagents and LPA carrier. The samples were compared to whole Qiagen setting control with their carrier by running on 2%Agarose with histogram showing percentage of bands intensities comparable to Qiagen kit. B RT-PCR of N-450 using MeV210 and MeV217 primers for RNA purified with in-house protocol comparable to Qiagen QIAamp viral RNA mini kit
Fig. 4
Fig. 4
RT-PCR of N-450 using MeV210 and MeV217 primers for RNA purified with our homemade kit solutions and carrier. The RT-PCR was done for RNA extracted after serial dilution of infected Vero cells. Amplicons with 626 bp were detected by 2% agarose gel electrophoresis. 50 bp generuler ladder (thermoscientific) was used as standard
See this image and copyright information in PMC

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