RNA extraction from tissue swabs using Ampure XP beads and spin filtration

First experiment performed 19/4/20 using the solutions published in Open Wet Ware

https://openwetware.org/wiki/SPRI_bead_binding_buffer

Recipe (DNA version)Final concentrationsPEG 8000 20% (w/v) Sodium chloride 2.5 M Tris base 10 mM Disodium EDTA 1 mM Tween 20 0.05% (v/v) HCl 3.36 mM Preparing 50 mL of 1X working solutionSodium chloride, 5 M 25.000 mL Nuclease-free water 3.582 mL Tris base, 1 M 0.500 mL Disodium EDTA, 0.1 M 0.500 mL HCl, 1 N 0.168 mL PEG 8000, 50% (w/v) 20.000 mL Tween 20, 10% (v/v) 0.250 mL

Recipe (RNA version)Final concentrationsPEG 8000 20% (w/v) Sodium chloride 2.5 M Trisodium citrate 1 mM Tween 20 0.05% (v/v) HCl 0.56 mM Preparing 50 mL of 1X working solutionSodium chloride, 5 M 25.000 mL Nuclease-free water 4.672 mL Trisodium citrate, 1 M 0.050 mL HCl, 1 N 0.028 mL PEG 8000, 50% (w/v) 20.000 mL Tween 20, 10% (v/v) 0.250 mL

Extractions carried out using Qiagen RNeasy as control, and magnetic and spin extractions in parallel from XP beads

Filter plates – Millipore Multiscreen Durapore 0.65um, low nucleic acid binding membrane. XP bead size is 1um so they do not traverse the membrane – no beads were visible in the wells after test spins.

Input – 2x tubes of inactivated MEFs used as starting material, spun down and lysed in 400ul RLT by vortexing and Qiashredder

50ul then extracted with RNeasy column as control and eluted into 30ul. Yield 217ng/ul, i.e. >6ug

2 x 50ul incubated for 10 mins with 100ul beads suspended in DNA buffer 2 x 50ul incubated for 10 mins with 100ul beads suspended in RNA buffer

o beads float a bit in the RNA buffer so have to be intermittently mixed One of each DNA & RNA extracted on the magnetic rack in the usual manner with 3x 80%

EtOH washes, 200ul each. o The beads were very clumpy, presumably due to the large amount of DNA present,

but they resuspended in water for elution into 30ul The other two were loaded into one well each of a filter plate and spun for 4 minutes at

4000rpm (3200 rcf) in the plate centrifuge over a collection plateo About half of the liquid spun through but due to overloading with excess nucleic

acids the filter clogged and so the remainder was pipetted off the top Washes carried out with 80% ethanol, 3 x 200ul, 1 minute each at 4000 rpm

o These spun through with no issues When the beads lost their sheen, they were assumed to be dry enough for elution

o One minute was sufficient Elution with 30ul water and a 3-minute incubation followed by 2-minute spin at full speed

over a hard-shell PCR plateo Not all of the water passed through the bead bed, probably due to the quantity of

beads and nucleic acid present so the column effectively cloggedo Beads spun through on their own without nucleic acid present didn’t do this

Yields:

1. RNeasy – 217ng/ul2. DNA buffer, magnet – 190ng/ul3. RNA buffer, magnet – 215ng/ul4. DNA buffer, plate – 65ng/ul5. RNA buffer, plate – 61ng/ul

Conclusions so far:1. RNA quality is excellent and will be very suitable for cDNA and assay2. The 2:1 ratio of bead buffer to RLT works well3. Spin plates work well and are fast and easy but unsuitable for large input quantities, need to

establish what the reasonable limit is to get quantitative elution4. The home-made buffers work well

Follow-up experiments

In a previous experiment (not shown) I determined that input of lysate containing less than 1ug of nucleic acids worked very reliably and extraction efficiency and consistency is excellent using 50ul of XP beads. I wanted to measure viral recovery from lysate so used a lentiviral transduction titration plate as a surrogate retroviral RNA source. The titre in transduction units is a 2-fold serial dilution in 6 steps from 32000 to 2000. I do not know what this corresponds to in terms of viral genome copy number, suitable control material and primers are on order.

Materials and EquipmentAmpure XP beads resuspended in RNA SPRI bead binding solution (sourced from Open Wet Ware website)

PEG 8000 20% (w/v) Sodium chloride 2.5 M Trisodium citrate 1 mM Tween 20 0.05% (v/v) HCl 0.56 mM

Millipore Multiscreen filter plates with Durapore membrane, 0.65um pore sizeQiagen RLT lysis bufferEppendorf 5810R benchtop centrifuge with plate carriersThermo AB-0765 0.8ml storage plates (waste collection)Eppendorf Twin Tec hard shell fully skirted 96 well PCR plates – eluate collectionSuperscript IV reverse transcriptaseDharmacon SMARTchoice promoter selection plate

RNA extraction1. 106 frozen cells (mouse embryo fibroblasts) spun down for 1 minute at 3000 rpm in

microfuge and resuspended in 700ul Qiagen RLT2. Complete lysis ensured by vortexing for 1 minute 3. Lysate divided into 2 sets of 7 x 50ul aliquots on a storage plate4. Lentiviral serial dilution added – 1ul/well, 6 wells leaving the last one blank5. Control set of aliquots extracted with Qiagen RNeasy kit and eluted with 30ul water6. Test set extracted with XP beads in RNA binding buffer as follows:

a. 50ul beads at 1x manufacturers supplied concentration diluted 1:1 with RNA binding buffer

b. 100ul of bead suspension added to each 50ul lysate and binding allowed to take place for 10 minutes with occasional vortexing

c. Binding mix applied to filter plate with multichannel pipette and plate placed securely on 0.8ml storage plate then spun for 1 minute at 4000 rpm in plate centrifuge, leaving beads evenly distributed over filter bed

d. 200ul of 80% ethanol applied to wells and plate spun for 1 minute at 4000 rpme. Wash repeated twice moref. Beads dried briefly – the surface takes on a matt appearance, about 1 minuteg. 30ul water added per well, plate incubated at room temp for 3 minutes

h. Plate placed on Twin Tec PCR plate for elution and spun for 1 minute at 4000 rpm

Yields were all close to 20ng/ul, i.e. 600ng/well No significant difference seen between Qiagen and XP

cDNA synthesis and qPCR1. 5ul template RNA converted to cDNA using superscript IV according to the manufacturer’s

instructions, final volume 20ul 2. Virus qPCR carried out with Clontech LTR primers and SYBR green reporter3. Control set repeated with PPIA Taqman probe PCR

a. Unfortunately I currently have no suitable mouse control that works with SYBR qPCR

PPIA Taqman assay

I did not expect amplification in the no RT control as the PPIA probe set spans exons, however the two sets of preps are clearly comparable and the Ampure spin plate preps are performing as well as the Qiagen extractions

-RT+RT

Ampure

Qiagen

-RT+RT

-ve

Lentivirus titration

There was significant DNA carryover with the no RT control amplifying on average 3 CTs later than the cDNA. I believe this is due to plasmid carried over from the transfections originally used to produce the virus.

The experiment was repeated with Qiagen Quantitect, which incorporates an initial DNA removal step, leading to a large improvement with a difference of 10 CTs between cDNA and no RT control and a consistent

Ongoing work The viral titre experiment is being repeated independently, with more replicates and newly

arrived controls Optimal operating windows for extraction to be determined in detail Post-elution DNase procedure to be worked up, to enable method usage with any brand of

reverse transcriptase