May 23, 2023
AAV viral DNA and whole RNA recovery for AAV pool experiments in rhesus macaque
- 1California Institute of Technology
Protocol Citation: Miguel Chuapoco 2023. AAV viral DNA and whole RNA recovery for AAV pool experiments in rhesus macaque. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4jo68lo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 08, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 81617
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020495
Abstract
This protocol outlines procedures to extract viral DNA and whole RNA from rhesus macaque tissue that had been treated with AAV in vivo
Protocol materials
UltraPure Distilled Water Invitrogen - Thermo FisherCatalog #10977-015
Step 7
RNase Cocktail™ Enzyme MixThermo FisherCatalog #AM2286
Step 8
UltraPure DNase/RNase-Free Distilled WaterThermo Fisher ScientificCatalog #10977023
Step 8.1
Q5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L
In 2 steps
GlycoBlue™ CoprecipitantThermo ScientificCatalog #AM9516
Step 5
Zymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014
In 3 steps
CutSmart Buffer - 5.0 mlNew England BiolabsCatalog #B7204S
Step 8
DNase I, Amplification GradeThermo FisherCatalog #18068015
Step 8.1
SuperScript™ III Reverse TranscriptaseThermo FisherCatalog #18080093
Step 8.3
TRIzol ReagentThermo Fisher ScientificCatalog #15596026
Step 1
NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) - 96 rxnsNew England BiolabsCatalog #E7600S
Step 16
UltraPure™ Low Melting Point AgaroseThermo Fisher ScientificCatalog #16520050
Step 17
Sodium Acetate 3M, pH 5.2Thermo ScientificCatalog #R1181
Step 5
SmaI - 2,000 unitsNew England BiolabsCatalog #R0141S
Step 8
DNA/RNA extraction
DNA/RNA extraction
3h 8m
3h 8m
Add 100 mg tissue sample (brain or liver) and 1 mL TRIzol reagent TRIzol ReagentThermo Fisher ScientificCatalog #15596026 to bead homogenizer tubes. Use prefilled tubes with 1.5 mm Zirconium beads or 2.8 mm stainless steel beads.
Equipment
Prefilled 2.0ml tubes, Zirconium Beads, 1.5mm Triple-Pure - High Impact, 50pk
NAME
Homogenizer tubes (1.5 mm Zirconium beads)
TYPE
Benchmark Scientific
BRAND
D1032-15
SKU
LINK
Equipment
Prefilled 2.0ml tubes, Stainless Steel, 2.8mm Acid Washed, 50pk
NAME
Homogenizer tubes (2.8 stainless steel)
TYPE
Benchmark Scientific
BRAND
D1033-28
SKU
LINK
Homogenize tissue in using the following settings:
- Speed: 5.0 m/s
- Time: 30 seconds
- Pause: 1 minute
- Cycles: 2
Incubate for 00:05:00 .
Equipment
BEADBUG 6, SIX POSITION HOMOGENIZER, 115V
NAME
Tissue homogenizer (6 position)
TYPE
Benchmark Scientific
BRAND
D1036
SKU
LINK
Equipment
BEADBLASTER 24 MICROTUBE HOMOGENIZER, 115V
NAME
Tissue Homogenizer (24 position)
TYPE
Benchmark Scientific
BRAND
D2400
SKU
LINK
Note
Samples can be stored at -20 ºC for up to year in TRIzol.
5m
Centrifuge the homogenizer tubes containing the TRIzol solution and homogenized tissue using the following parameters: 12000 x g, 4°C, 00:05:00 . Transfer the supernatant to a new tube (microcentrifuge tube or similar).
Equipment
Centrifuge 5425/5425 R - Microcentrifuge
NAME
Refrigerated centrifuge
TYPE
Eppendorf
BRAND
2231000909
SKU
LINK
Equipment
DNA LoBind® Tubes
NAME
Microcentrifuge tubes
TYPE
Eppendorf
BRAND
022431021
SKU
LINK
5m
Add 200 µL chloroform to each tube for every 1 mL TRIzol used for lysis, vortex briefly, and incubate for 00:03:00 .
3m
Add 1 equivalent volume of isopropanol, 1/10 volume of sodium acetate, and co-precipitant (e.g. 500 µL isopropanol , 50 µL sodium acetate , 2-3 µL co-precipitant ) and vortex briefly. Incubate for 00:10:00 .
Sodium Acetate 3M, pH 5.2Thermo ScientificCatalog #R1181
GlycoBlue™ CoprecipitantThermo ScientificCatalog #AM9516
10m
Centrifuge 12000 x g, 4°C, 00:10:00 to pellet nucleic acids. Discard supernatant and wash pellet with 1 mL 75% ethanol . Centrifuge again 7500 x g, 4°C, 00:05:00 and discard supernatant.
15m
Air dry pellet and resuspend in 84 µL PCR clean water
UltraPure Distilled Water Invitrogen - Thermo FisherCatalog #10977-015
To isolate DNA, treat half of the sample with RNase. Remove RNA by digestion with 1.5 µL RNase cocktail and digest with 1.5 µL SmaI . Supplement reaction with 5 µL CutSmart . Incubate at Room temperature for 2-3 hours and 37 °C overnight .
Purify with Zymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014
RNase Cocktail™ Enzyme MixThermo FisherCatalog #AM2286
SmaI - 2,000 unitsNew England BiolabsCatalog #R0141S
CutSmart Buffer - 5.0 mlNew England BiolabsCatalog #B7204S
To obtain cDNA, take 1 µg RNA from sample (measured by high sensitivity RNA Qubit) and treat with DNase I, Amplification GradeThermo FisherCatalog #18068015
Combine 1 µg RNA with 1 µL 10X DNase I reaction buffer , 1 µL DNase I , and UltraPure DNase/RNase-Free Distilled WaterThermo Fisher ScientificCatalog #10977023 to 10 uL.
Incubate reaction Room temperature 00:15:00
15m
Inactivate DNase I by adding 1 µL 25 mM EDTA . Heat 65 °C 00:15:00
15m
To convert DNase I treated RNA to cDNA, take 1-5 µL sample and combine with 1 µL oligo(dT) , 1 µL dNTP and fill to 10 µL using UltraPure water.
SuperScript™ III Reverse TranscriptaseThermo FisherCatalog #18080093
Incubate 65 °C 00:05:00 and place on ice.
5m
Prepare cDNA synthesis mix according to manufacturer's specifications. Add 10 µL cDNA synthesis mix to each RNA primer mixture and mix by gently flicking the tubes.
Incubate as follows:
- 50 °C 00:50:00
- 25 °C 00:10:00
- 50 °C 00:50:00
- 85 °C 00:05:00
1h 55m
Store samples at -80 °C until ready to use.
PCR amplification
PCR amplification
3h 8m
3h 8m
Use Zymo DNA purification of PCR product according to manufacturer's suggested protocol.Zymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014
Dilute PCR product 1:100 and use as template for an additional round of PCR amplification around the variable region with primers containing Read1 and Read2 sequences by 10 cycles of 98 °C for 00:00:10 , 60 °C for 00:00:30 , and 72 °C for 00:00:10 using Q5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L and the following primers:
- Forward: 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGcgatgttccagattacgcttgag -3'
- Reverse: 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGattttgtaatccagaggttgattatcg - 3'
50s
Use Zymo DNA purification of PCR product according to manufacturer's suggested protocol.Zymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014
Append Illumina flow cell adapters and unique indices by PCR amplification with NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) - 96 rxnsNew England BiolabsCatalog #E7600S
by 10 cycles of 98 °C for 00:00:10 , 59 °C for 00:00:30 , and 72 °C for 00:00:10 using Q5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L
50s
Run PCR products on a freshly-prepared 2% UltraPure™ Low Melting Point AgaroseThermo Fisher ScientificCatalog #16520050 gel and gel purify amplified 200 bp PCR product.