Chapter 3: PROPAGATION AND EXTRACTION OF AHS VIRAL RNA
3.2 Materials and Methods
3.2.3 AHSV RNA Extraction
The extraction of viral RNA from the egg yolk and from the cell culture supernatants was achieved using TRIzol® LS Reagent (Invitrogen, Carlsbad, USA). Various variations to the standard protocol of Invitrogen (Carlsbad, USA) were used to extract viral RNA from the egg yolk as the standard protocol resulted in no RNA being extracted (Additional or amended steps for the yolk extraction are shown in italics).
3.2.3.1 Extraction from egg yolk
3.2.3.1.1 Homogenisation
250 µL of egg yolk sample and 750 µL of TRIzol® LS Reagent were combined in a microcentrifuge tube to achieve a minimum ratio of one part sample to three parts TRIzol® LS Reagent. The sample was homogenised by passing several times through a Gilson Pipetman® P1000 pipette. The initial yolk sample was diluted 1:1 with DEPC- water and compared to non-diluted samples (Invitrogen, 2007; Watson, Personal Communication). Homogenisation was also achieved using a 21G needle (Watson, Personal Communication).
An extra centrifugation step was added after homogenisation to remove any insoluble material from the yolk. The homogenised sample was centrifuged at 12,000 × g for 10 minutes at 4°C. Three phases resulted and all but the top, fatty layer were removed for downstream steps (Invitrogen, 2007).
3.2.3.1.2 Phase Separation
The homogenised samples were incubated for 5 minutes at room temperature (15 to 30°C). 150 µL of chloroform was added at a ratio of 0.2 mL of chloroform per 1 mL of TRIzol® LS Reagent. The tubes were capped securely and vigorously shaken for 15
seconds followed by an incubation period of a few minutes at room temperature (15 to 30°C). The samples were subsequently centrifuged at 12,000 × g for 15 minutes at 4°C in an Eppendorf® Microcentrifuge 5415 R.
3.2.3.1.3 RNA Precipitation
After centrifugation, three layers are apparent: an upper aqueous phase, an interphase and a bottom phenol chloroform phase. RNA in the upper aqueous phase was removed and transferred to a fresh tube. The RNA was precipitated from the aqueous phase by mixing with 375 µL of isopropyl alcohol to achieve a ratio of 0.5 mL of isopropyl alcohol per 1 mL of TRIzol® LS Reagent used for the initial homogenization.
The samples were incubated at room temperature (15 to 25°C) for 10 minutes and subsequently centrifuged at no more than 12,000 × g for 10 minutes at 4°C.
Additionally, pure isopropyl alcohol was compared to a 1:1 mix of isopropyl alcohol and 1.2 M NaCl (Invitrogen, 2007; Watson, Personal Communication).
3.2.3.1.4 RNA Wash
The supernatant was poured off and the RNA pellet was washed once with 950 µL 75% (v/v) ethanol, adding at least 1 mL of 75% ethanol per 1 mL of TRIzol® LS Reagent used for the initial homogenization. The sample was mixed by vortexing briefly and centrifuged at no more than 7,500 × g for 5 minutes at 4°C.
3.2.3.1.5 Redissolving the RNA
The supernatant was poured off and the tubes returned to the centrifuge and spun down briefly to collect the last drops of ethanol. The tubes were then carefully aspirated and the RNA pellet left to air-dry for 5 minutes. The pellet was then dissolved in 40 µL of DEPC-treated water and incubated at 55-60°C for 10 minutes.
Standard protocols failed to yield detectable results and it was therefore adjusted to include some additional steps:
The additional steps resulted in four different RNA preparations that were compared (Table 3.3). In both situations, 4 µL of the extractions with 10 µL of formaldehyde loading buffer were run on ethidium bromide (0.5 µg/mL) containing 1.2 % (w/v) agarose gels in 0.5 × TBE.
Table 3.3: Summary of additional procedures in the TRIzol® LS extraction of AHS viral RNA from ECE
Number Treatment
1 Undiluted yolk as initial sample RNA precipitated with IPA only
2 Undiluted yolk as initial sample
RNA precipitated with IPA and 1.2 M NaCl
3 1:1 yolk : water as initial sample RNA precipitated with IPA only
4 1:1 yolk : water as initial sample
RNA precipitated with IPA and 1.2 M NaCl
3.2.3.2 Extraction from cell culture
3.2.3.2.1 1. Homogenisation
1 mL aliquots for each serotype and control were thawed and transferred to a 15 mL Falcon™ tube (BD Biosciences, San Jose, USA). 3 mL of TRIzol® LS was added to achieve a 1:3 ratio of sample to reagent. The solution was homogenised by pipetting several times.
3.2.3.2.2 Phase Separation
To achieve the required phase separation, the homogenised samples were incubated at room temperature (15-25°C) for at least 60 minutes. 800 µL of chloroform was added and shaken vigorously by hand for 15 seconds. The shaken samples were incubated again at room temperature for 10-15 minutes and centrifuged at 12,000 × g for 15 mins at 4°C in a Beckman-Coulter Avanti® J-26XP centrifuge (JA-10 rotor).
3.2.3.2.3 RNA Precipitation
The solution separated into a lower red-pink phase, a white opaque interphase and an upper clear aqueous phase. RNA remains exclusively in the aqueous phase (Invitrogen, 2007). The upper aqueous phase was transferred to a new tube containing 750 µL of a 1.2 M sodium chloride/0.8 M sodium citrate sterile solution and 750 µL of ice-cold isopropyl alcohol. The tubes were inverted several times and incubated at room temperature for 10-15 minutes followed by a centrifugation at 12,000 × g for 10 minutes at 4°C.
3.2.3.2.4 RNA Wash
The resultant supernatant was discarded and the translucent pellet was washed by adding 4 mL of 75% ethanol in DEPC-treated water. The tubes were vortexed for 30 seconds and centrifuged at 7,500 × g for 5 minutes at 4°C. The supernatant was discarded and the pellet and remaining ethanol was aspirated and transferred to a 1.5 mL micro-centrifuge tube. The tubes were centrifuged at 7,500 × g for 5 minutes at 4°C in an Eppendorf® Microcentrifuge 5415 R. The remaining ethanol was carefully aspirated and the pellet air-dried for 5 minutes.
3.2.3.2.5 Redissolving the RNA
The pellet was subsequently re-suspended in 20 µL of DEPC-treated water and incubated at 55-60°C for 10 minutes. Absorbance readings were taken on the suspension after extraction with TRIzol® using a Thermo Scientific NanoDrop 1000.