Abstract
High quality RNA is critical for molecular and viral diagnostic applications. In case of citrus, polyphenol and polysaccharide rich content hinders many RNA isolation methods causing low quality RNA. These shortcomings can affect the Citrus tristeza virus (CTV) detection which is responsible to cause worldwide destruction of citrus orchards. Thus, in this study we analysed the RNA quality, quantity and integrity of different commercial and conventional methods. Commercial methods include TRIzol and kit-based method (PureLink RNA kit), while Cetyl trimethylammonium bromide (CTAB) and SDS/ Phenol (TENS-PCI) based method were optimized as conventional methods. For further validation, amplification potential was evaluated by performing Reverse transcriptase polymerase chain reaction (RT PCR) of CTV coat protein (p25) and mRNA internal control (nad5) genes to affirm RNA quality. Results indicated poor RNA recovery from TRIzol method with no successful amplification at RT PCR. Contrastingly, the modified conventional methods indicated RNA quality and purity for nucleic acids at Absorbance 260/280 (1.8–2.1) and Absorbance 260/230 (1.8–2.2) proving drastic improvement in RNA quality with reproducible amplification results. These results were further demonstrated with stored samples of different citrus varieties which indicated poor RNA integrity but successful RT PCR amplification. Consequently, our results may provide a brief comparison between isolation procedures by providing reference values to determine RNA purity requirements prior to RT PCR and help to determine rapidity, reproducibility and yield for each isolation method.
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Abbreviations
- RNA:
-
Ribonucleic acid
- mRNA:
-
Messenger ribonucleic acid
- CTV:
-
Citrus tristeza virus
- CTAB:
-
Cetyl trimethylammonium bromide
- TENS-PCI:
-
SDS/Phenol method
- nad5 :
-
NADH-ubiquinone oxidoreductase
- p25::
-
Major coat protein gene (CTV)
- RT PCR:
-
Reverse transcriptase polymerase chain reaction
- A260/280:
-
Absorbance at 260/ Absorbance at 280
- A260/230:
-
Absorbance at 260/ Absorbance at 230
- DEPC:
-
Diethyl pyrocarbonate
- C.reticulata :
-
Citrus reticulata
- C.paradise :
-
Citrus paradise
- C. sinensis :
-
Citrus sinensis
- C. limetta :
-
Citrus limetta
- TBE:
-
Tris/Borate/ Ethylenediaminetetraacetic acid
- ANOVA:
-
Analysis of variance
- LSD:
-
Least significant difference test
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Nesheman Huma and Shagufta Sahar contributed to the study conception and design. Material preparation, data collection and analysis was performed by Nesheman Huma. Saddia Galani have checked the requirements and provided guidance. The first draft of the manuscript was written by Nesheman Huma. All the authors have checked and commented on the manuscript. The research was supervised by Dr. Saddia Galani. All authors have read and approved this manuscript.
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Huma, N., Sahar, S., Iftikhar, Y. et al. RNA isolation efficacy of commercial and modified conventional methods for Citrus tristeza virus and mRNA internal control amplification. Biologia 75, 1195–1202 (2020). https://doi.org/10.2478/s11756-019-00405-w
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DOI: https://doi.org/10.2478/s11756-019-00405-w