TITLE: Sterivex RNA extraction
AUTHORS: Ariel Rabines, Rob Lampe, Andrew Allen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A mostly automated protocol for extraction of total RNA from seawater filtered onto a Sterivex filter (Cat. No. SVGP0150). Reagents come from the Macherey-Nagel NucleoMag RNA kit (Cat. No. 744350). Automated liquid handling is performed on an eppendorf EpMotion 5075t with multi-channel pipettes. This protocol assumes that the Sterivex is sealed with tube sealant on the male end and a male-luer lock plug on the female end. Using this protocol, we routinely extract ~80 samples at a time.</div><div class = "text-block">The protocol has been modified to increase to an 800 µL starting volume. This normally results in less Lysis Buffer MR1, Binding Buffer MR2, and Reducing Agent TCEP supplied than is needed. Extra reagents can and should be purchased separately. </div><div class = "text-block"><span>The kit manual can be found here: </span><a href="https://www.mn-net.com/media/pdf/23/c2/23/Instruction-NucleoMag-RNA.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.mn-net.com/media/pdf/23/c2/23/Instruction-NucleoMag-RNA.pdf</span></a></div><div class = "text-block"><span>Additional notes from the manufacturer on using the kit with the epMotion can be found here: </span><a href="https://www.mn-net.com/media/pdf/bd/7e/53/AN-NucleoMag-RNA-epMotion5075.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.mn-net.com/media/pdf/bd/7e/53/AN-NucleoMag-RNA-epMotion5075.pdf</span></a><span> . Note that we have several additional modifications to their protocol.</span></div><div class = "text-block">The epMotion program is attached here.</div></div>

[STEPS]
?. [Lysis]
Keep sterivex on dry ice. In each, remove luer plug and pipette the per sample volume prepared in step 1 into each. For example, if 800 µL MR1 + 14 µL TCEP + 1 µL internal standards were used, add . Dispense quickly as the lysis buffer will freeze upon contact. Replug with luer lock. Place luer-side down into 1.5 mL tube rack.
815 µl
?. [Lysis]
Incubate at   and shake gently for  .
33 °C
?. Vortex for  using adapter for 5 mL tubes.
?. Transfer lysate to sterile 1.5 mL tubes labeled with extraction numbers.
?. Label each sterviex with its extraction number.
?. Pop the lid with the male end off the end of the sterivex using the tube cutters. The tube cutters will fit in the lip between the lid and the rest of the unit. Apply pressure at an upward angle and it should release. Continue to cut around the top if needed.
?. Rub the filter on the inside of the sterviex to release as much of the lysis buffer and material as possible. Then pipette as much liquid as possible from inside the sterviex into the corresponding 1.5 mL tube.
?. Clear lysate by centrifugaion at  .
Centrifuge: 5600 34, 6 min
This is a good time to prepare your buffers. The epMotion will calculate your buffer volumes depending on the number of samples. Add a few mL more than is requested and enter that as the volume supplied. We premix the beads and Binding Buffer MR2 at a ratio of 28 µL beads:772 µL MR2. Note that this keeps the bead amount the same as specified in the manual while increasing the amount of MR2.
?. Transfer to another set of numbered 1.5 mL tubes. Remove as much supernatent as possible without disturbing the pellet.
?. Align 1.5 mL tubes in rack in plate position. Set adjustable spacer multichannel pipette to  and transfer no more than  into the deep well plate.
800 µl
800 µl
?. Transfer deep well plate to the epMotion and run program. The final elution is always into a twin.tec LoBind PCR plate (see materials).
Reservoir rack layout (left to right):MR2 with beadsMR3MR4MR4MR5MR2 without beadsDNase
Step 4 in the program stops it. At this point with a P1000 multichannel pipette, throughly pipette up and down to mix the bead solution then continue the run.
?. [Prepare working solutions]
rDNase working solution: Add  of RNase-free H2O to the rDNase vials. Incubate for 1 min at room temperature. Gently swirl to completely dissolve. Do not mix vigourously. This can be stored at  for up to 6 months. Do not freeze / thaw more than three times.
800 µl
-20 °C
?. [Prepare working solutions]
rDNase reaction mixture: From epMotion program, quantify how much rDNase you will need for your extraction. Gently mix at the following ratio:   Reaction Buffer for rDNase:   rDNase working solution.
276 µl
24 µl
?. [Prepare working solutions]
Reducing Agent TCEP: Add   of RNase-free H2O to the TCEP vial and incubate for several minutes at room temperature. Mix the vial to dissolve the TCEP completely. Store dissolved TCEP at .
750 µl
-20 °C
?. Quantification: Make a 1:10 dilution plate ( total volume) using the elution buffer MR5 (RNase-free H2O). Quantify the dilution plate with RiboGreen assay and use that to calculate the concentration and total RNA in the original plate.
20 µl
?. Quality: Analyze 1 µL on Agilent Tapestation with RNA screentape.
?. [Prepare working solutions]
Lysis Buffer (N = number of samples plus extra for pipetting; X = quantity of internal standard mix (usually 1-2.5 μL):In sterile container, add   x N of Lysis Buffer MR1. Add  x N TCEP. Add X x N μL of internal standard mix. Mix well
800 µl
14 µl